Nicked or gapped nucleic acid molecules and uses thereof

ABSTRACT

The present disclosure provides meroduplex (nicked or gapped) ribonucleic acid molecules (mdRNA) that decreases or silences target gene expression. An mdRNA of this disclosure comprises at least three strands that combine to form at least two non-overlapping double-stranded regions separated by a nick or gap wherein one strand is complementary to a target gene RNA. In addition, the meroduplex may have one or more modifications or substitutions, such as nucleotide base, sugar, terminal cap structure, internucleotide linkage, or any combination of such modifications. Also provided are methods of decreasing expression of a target gene in a cell or in a subject to treat a disease related to altered expression of a target gene.

TECHNICAL FIELD

The present disclosure provides double-stranded nucleic acid moleculescapable of gene silencing and, more specifically, a nicked or gappeddouble-stranded RNA (dsRNA) comprising at least three strands thatdecreases expression of a target gene by, for example, RNA interferenceand uses of such dsRNA to treat or prevent disorders associated withexpression of the target gene or genes affected by the target gene.

BACKGROUND

RNA interference (RNAi) refers to the cellular process of sequencespecific, post-transcriptional gene silencing in animals mediated bysmall inhibitory nucleic acid molecules, such as a double-stranded RNA(dsRNA) that is homologous to a portion of a targeted messenger RNA(Fire et al., Nature 391:806, 1998; Hamilton et al., Science286:950-951, 1999). RNAi has been observed in a variety of organisms,including mammalians (Fire et al., Nature 391:806, 1998; Bahramian andZarbl, Mol. Cell. Biol. 19:274-283, 1999; Wianny and Goetz, Nature CellBiol. 2:70, 1999). RNAi can be induced by introducing an exogenoussynthetic 21-nucleotide RNA duplex into cultured mammalian cells(Elbashir et al., Nature 411:494, 2001a).

The mechanism by which dsRNA mediates targeted gene-silencing can bedescribed as involving two steps. The first step involves degradation oflong dsRNAs by a ribonuclease III-like enzyme, referred to as Dicer,into short interfering RNAs (siRNAs) having from 21 to 23 nucleotideswith double-stranded regions of about 19 base pairs (Berstein et al.,Nature 409:363, 2001; Elbashir et al., Genes Dev. 15:188, 2001b; and Kimet al., Nature Biotech. 23(2):222, 2005). The second step of RNAigene-silencing involves activation of a multi-component nuclease havingone strand (guide or antisense strand) from the siRNA and an Argonauteprotein to form an RNA-induced silencing complex (“RISC”) (Elbashir etal., Genes Dev. 15:188, 2001). Argonaute initially associates with adouble-stranded siRNA and then endonucleolytically cleaves thenon-incorporated strand (passenger or sense strand) to facilitate itsrelease due to resulting thermodynamic instability of the cleaved duplex(Leuschner et al., EMBO 7:314, 2006). The guide strand in the activatedRISC binds to a complementary target mRNA and cleaves the mRNA topromote gene silencing. Cleavage of the target RNA occurs in the middleof the target region that is complementary to the guide strand (Elbashiret al., 2001b).

Target specific gene silencing can be achieved by exogenously addingsiRNA, but non-specific silencing of non-targeted genes (referred to asoff-target effects) can be a challenge (see, e.g., Jackson et al., Nat.Biotechnol. 21:635, 2003; Du et al., Nucleic Acids Res. 33:1671, 2005.Hence, there remains a need in the art for alternative dsRNA moleculesand methods to mediate gene silencing. The present disclosure meets suchneeds, and further provides other related advantages.

BRIEF SUMMARY

The present disclosure provides dsRNA molecules comprising at leastthree strands, designated herein as A, S1 and S2 (A:S1S2), wherein S1and S2 are complementary to, and form base pairs (bp) with,non-overlapping regions of A. Thus, for siRNA molecules describedherein; the double-stranded region formed by the annealing of S1 and Ais distinct from the double-stranded region formed by the annealing ofS2 and A. An A:S1 duplex may be separated from an A:S2 duplex by a “gap”resulting from at least one unpaired nucleotide in the A strand that ispositioned between the A:S1 duplex and the A:S2 duplex and that isdistinct from any one or more unpaired nucleotide at the 3′ end ofeither or both of the A, S1, and/or S2 strand. Alternatively, an A:S1duplex may be separated from an A:S2 duplex by a “nick” such that thereare no unpaired nucleotides in the A strand that are positioned betweenthe A:S1 duplex and the A:S2 duplex such that the only unpairednucleotide, if any, is at the 3′ end of either or both of the A, S1,and/or S2 strand.

In one aspect, the instant disclosure provides a meroduplex RNA (mdRNA)molecule, comprising a first strand that is complementary to a targetRNA, and a second strand and a third strand that are each complementaryto non-overlapping regions of the first strand, wherein the secondstrand and third strands can anneal with the first strand to form atleast two double-stranded regions separated by a gap of up to 10nucleotides, and wherein (a) at least one double-stranded region is fromabout 5 base pairs up to 13 base pairs, or (b) the double-strandedregions combined total about 15 base pairs to about 40 base pairs andthe mdRNA molecule comprises blunt ends. In certain embodiments, thefirst strand is about 15 to about 40 nucleotides in length, and thesecond and third strands are each, individually, about 5 to about 20nucleotides, wherein the combined length of the second and third strandsis about 15 nucleotides to about 40 nucleotides. In other embodiments,the mdRNA is a RISC activator (e.g., the first strand has about 15nucleotides to about 25 nucleotides) or a Dicer substrate (e.g., thefirst strand has about 26 nucleotides to about 40 nucleotides). In someembodiments, the gap comprises at least one to ten unpaired nucleotidesin the first strand positioned between the double-stranded regionsformed by the second and third strands when annealed to the firststrand, or the gap comprises a nick. In certain embodiments, the nick orgap is located 10 nucleotides from the 5′-end of the first (antisense)strand or at the Argonaute cleavage site. In another embodiment, themeroduplex nick or gap is positioned such that the thermal stability ismaximized for the first and second strand duplex and for the first andthird strand duplex as compared to the thermal stability of suchmeroduplexes having a nick or gap in a different position.

In another aspect, the instant disclosure provides an mdRNA moleculehaving a first strand that is complementary to target RNA, and a secondstrand and a third strand that is each complementary to non-overlappingregions of the first strand, wherein the second strand and third strandscan anneal with the first strand to form at least two double-strandedregions separated by a gap of up to 10 nucleotides, and wherein (a) atleast one double-stranded region is from about 5 base pairs up to 13base pairs, or (b) the double-stranded regions combined total about 15base pairs to about 40 base pairs and the mdRNA molecule comprises bluntends; and wherein at least one pyrimidine of the mdRNA comprises apyrimidine nucleoside according to Formula I or II:

wherein R¹ and R² are each independently a —H, —OH, —OCH₃, —OCH₂OCH₂CH₃,—OCH₂CH₂OCH₃, halogen, substituted or unsubstituted C₁-C₁₀ alkyl,alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino,aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl,trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted —O-allyl,—O—CH₂CH═CH₂, —O—CH═CHCH₃, substituted or unsubstituted C₂-C₁₀ alkynyl,carbamoyl, carbamyl, carboxy, carbonylamino, substituted orunsubstituted aryl, substituted or unsubstituted aralkyl, —NH₂, —NO₂,—C≡N, or heterocyclo group; R³ and R⁴ are each independently a hydroxyl,a protected hydroxyl, a phosphate, or an internucleoside linking group;and R⁵ and R⁸ are independently O or S. In certain embodiments, at leastone nucleoside is according to Formula I and in which R¹ is methyl andR² is —OH. In certain related embodiments, at least one uridine of thedsRNA molecule is replaced with a nucleoside according to Formula I inwhich R¹ is methyl and R² is —OH, or R¹ is methyl, R² is —OH, and R⁸ isS. In some embodiments, the at least one R¹ is a C₁-C₅ alkyl, such asmethyl. In some embodiments, at least one R² is selected from2′-O—(C₁-C₅) alkyl, 2′-O-methyl, 2′-OCH₂OCH₂CH₃, 2′-OCH₂CH₂OCH₃,2′-O-allyl, or fluoro. In some embodiments, at least one pyrimidinenucleoside of the mdRNA molecule is a locked nucleic acid (LNA) in theform of a bicyclic sugar, wherein R² is oxygen, and the 2′-O and 4′-Cform an oxymethylene bridge on the same ribose ring (e.g., a5-methyluridine LNA) or is a G clamp. In other embodiments, one or moreof the nucleosides are according to Formula I in which R¹ is methyl andR² is a 2′-O—(C₁-C₅) alkyl, such as 2′-O-methyl. In some embodiments,the gap comprises at least one unpaired nucleotide in the first strandpositioned between the double-stranded regions formed by the second andthird strands when annealed to the first strand, or the gap comprises anick. In certain embodiments, the nick or gap is located 10 nucleotidesfrom the 5′-end of the first strand or at the Argonaute cleavage site.In another embodiment, the meroduplex nick or gap is positioned suchthat the thermal stability is maximized for the first and second strandduplex and for the first and third strand duplex as compared to thethermal stability of such meroduplexes having a nick or gap in adifferent position.

Compositions and methods disclosed herein are useful for reducingexpression of a target gene, or one or more genes that are a part of thetarget gene family, in a cell or to treating or preventing diseases ordisorders associated with expression of one or more target gene familymembers, such as hyperproliferative disorders (e.g., cancer),inflammatory conditions (e.g., arthritis), respiratory disease,pulmonary disease, cardiovascular disease, autoimmune disease, allergicdisorders, neurologic disease, infectious disease (e.g., viralinfection, such as influenza), renal disease, transplant rejection, orany other disease or condition that responds to modulation of a targetgene or gene family.

In certain embodiments, dsRNA of the present disclosure comprise, insum, between about 15 base-pairs and about 40 base-pairs; or betweenabout 18 and about 35 base-pairs; or between about 20 and 30 base-pairs;or 21, 22, 23, 24, 25, 26, 27, 28, or 29 base-pairs. Within certainembodiments, the siRNA may, optionally, comprise a single-strand3′-overhang of between 1 nucleotide and 5 nucleotides. In particularembodiments, such a single-strand 3′-overhang is 1, 2, 3, or 4nucleotides.

In another aspect, dsRNA of the present disclosure comprise either an Asense strand or an A antisense strand wherein the length of the A strandis between about 15 nucleotides and about 50 nucleotides; or the lengthof the A strand is between about 18 nucleotides and about 40nucleotides; or the length of the A strand is between about 20nucleotides and about 32 nucleotides; or the length of the A strand is21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides.

In another aspect, siRNA of the present disclosure additionally comprisetwo or more S strands, designated herein, for example, as S1 and S2,wherein each S strand is complementary to a non-overlapping region of acognate A strand and wherein a first S strand (S1) is separated from asecond S strand (S2) by a nick or a one or more nucleotide gap.Depending upon whether the cognate A strand is a sense strand or anantisense strand, each S strands will be either an antisense strand or asense strand, respectively. Each S strand (S1, S2, etc.) describedherein is, independently, between about 1 nucleotide and about 25nucleotides in length; more typically between about 4 nucleotides andabout 20 nucleotides in length; still more typically between about 5nucleotides and about 16 nucleotides in length; most typically 6, 7, 8,9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

Depending upon the precise application contemplated, a first S strand(S1) may be separated from a second S strand (S2) by a nick or by a gap.In those embodiments wherein S1 and S2 are separated by a gap, the gapis between about one nucleotide and about 25 nucleotides; or betweenabout one nucleotide and about 15 nucleotides; or between about onenucleotide and about 10 nucleotides; or the gap is 1, 2, 3, 4, 5, 6, 7,8, or 9 nucleotide(s). Each S strand may, independently, terminate witha 5′ hydroxyl (i.e., 5′-OH) or may terminate with a 5′ phosphate group(i.e., 5′-PO₄).

In any of the aspects of this disclosure, there are provided mdRNAmolecules having a 5-methyluridine (ribothymidine) or a2-thioribothymidine in place of at least one uridine on the first,second, or third strand, or in place of each and every uridine on thefirst, second, or third strand. In further embodiments, the mdRNA maycomprise any one of 5-methyluridine (ribothymidine),2-thioribothymidine, deoxyuridine, locked nucleic acid (LNA) molecule,sugar modified with 2′-Omethyl, or G clamp, or any combination thereof.In certain embodiments, the mdRNA molecule comprises a 2′-sugarsubstitution, such as a 2′-O-methyl, 2′-O-methoxyethyl,2′-O-2-methoxyethyl, 2′-O-allyl, or halogen (e.g., 2′-fluoro). Incertain embodiments, the mdRNA molecule further comprises at least oneterminal cap substituent on one or both ends of the first strand, secondstrand, or third strand, such as independently an alkyl, abasic, deoxyabasic, glyceryl, dinucleotide, acyclic nucleotide, or inverteddeoxynucleotide moiety. In other embodiments, the mdRNA molecule furthercomprises at least one modified internucleoside linkage, such asindependently a phosphorothioate, chiral phosphorothioate,phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methylphosphonate, alkyl phosphonate, 3′-alkylene phosphonate, 5′-alkylenephosphonate, chiral phosphonate, phosphonoacetate, thiophosphonoacetate,phosphinate, phosphoramidate, 3′-amino phosphoramidate,aminoalkylphosphoramidate, thionophosphoramidate,thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate, orboranophosphate linkage.

In any of the aspects of this disclosure, some embodiments provide anmdRNA comprising an overhang of one to four nucleotides on at least one3′-end that is not part of the gap, such as at least onedeoxyribonucleotide or two deoxyribonucleotides (e.g., thymidine). Insome embodiments, at least one or two 5′-terminal ribonucleotide of thesecond strand within the double-stranded region comprises a 2′-sugarsubstitution. In related embodiments, at least one or two 5′-terminalribonucleotide of the first strand within the double-stranded regioncomprises a 2′-sugar substitution. In other related embodiments, atleast one or two 5′-terminal ribonucleotide of the second strand and atleast one or two 5′-terminal ribonucleotide of the first strand withinthe double-stranded regions comprise independent 2′-sugar substitutions.In other embodiments, the mdRNA molecule comprises at least three5-methyluridines within at least one double-stranded region. In someembodiments, the mdRNA molecule has a blunt end at one or both ends. Inother embodiments, the 5′-terminal of the third strand is a hydroxyl ora phosphate.

It will be understood that methods of the present disclosure do notrequire a priori knowledge of the nucleotide sequence of every possiblegene variant(s) targeted by the gapped or nicked dsRNA. Initially, thenucleotide sequence of the siRNA may be selected from a conserved regionof the target gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows knockdown activity for RISC activator lacZ dsRNA (21nucleotide sense strand/21 nucleotide antisense strand; 21/21), Dicersubstrate lacZ dsRNA (25 nucleotide sense strand/27 nucleotide antisensestrand; 25/27), and meroduplex lacZ mdRNA (13 nucleotide sense strandand 11 nucleotide sense strand/27 nucleotide antisense strand; 13,11/27—the sense strand is missing one nucleotide so that a singlenucleotide gap is left between the 13 nucleotide and 11 nucleotide sensestrands when annealed to the 27 nucleotide antisense strand. Knockdownactivities were normalized to a Qneg control dsRNA and presented as anormalized value of Qneg (i.e., Qneg represents 100% or “normal” geneexpression levels). A smaller value indicates a greater knockdowneffect.

FIG. 2 shows knockdown activity of a RISC activator influenza dsRNAG1498 (21/21) and nicked dsRNA (10, 11/21) at 100 nM. The “wt”designation indicates an unsubstituted RNA molecule; “rT” indicates RNAhaving each uridine substituted with a ribothymidine; and “p” indicatesthat the 5′-nucleotide of that strand was phosphorylated. The 21nucleotide sense and antisense strands of G1498 were individually nickedbetween the nucleotides 10 and 11 as measured from the 5′-end, and isreferred to as 11, 10/21 and 21/10, 11, respectively. The G1498 singlestranded 21 nucleotide antisense strand alone (designated AS-only) wasused as a control.

FIG. 3 shows knockdown activity of a lacZ dicer substrate (25/27) havinga nick in one of each of positions 8 to 14 and a one nucleotide gap atposition 13 of the sense strand (counted from the 5′-end). A dideoxyguanosine (ddG) was incorporated at the 5′-end of the 3′-most strand ofthe nicked or gapped sense sequence at position 13.

FIG. 4 shows knockdown activity of a dicer substrate influenza dsRNAG1498DS (25/27) and this sequence nicked at one of each of positions 8to 14 of the sense strand, and shows the activity of these nickedmolecules that are also phosphorylated or have a locked nucleic acidsubstitution.

FIG. 5 shows a dose dependent knockdown activity a dicer substrateinfluenza dsRNA G1498DS (25/27) and this sequence nicked at position 13of the sense strand.

FIG. 6 shows knockdown activity of a dicer substrate influenza dsRNAG1498DS having a nick or a gap of one to six nucleotides that begins atany one of positions 8 to 12 of the sense strand.

FIG. 7 shows knockdown activity of a LacZ RISC dsRNA having a nick or agap of one to six nucleotides that begins at any one of positions 8 to14 of the sense strand.

FIG. 8 shows knockdown activity of an influenza RISC dsRNA having a nickat any one of positions 8 to 14 of the sense strand and further havingone or two locked nucleic acids per sense strand.

FIG. 9 shows knockdown activity of a LacZ dicer substrate dsRNA having anick at any one of positions 8 to 14 of the sense strand as compared tothe same nicked dicer substrates but having a locked nucleic acidsubstitution.

FIG. 10 shows the dose-dependent reduction in WSN influenza viral titersusing influenza specific mdRNA as measured by TCID₅₀.

FIG. 11 shows the percent knockdown in influenza viral titers usinginfluenza specific mdRNA against influenza strain WSN.

FIG. 12 shows the in vivo reduction in PR8 influenza viral titers usinginfluenza specific mdRNA as measured by TCID₅₀.

DETAILED DESCRIPTION OF THE DISCLOSURE

The instant disclosure provides gapped double-stranded RNA (dsRNA)comprising at least three strands that is a suitable substrate for Diceror for association with RISC and, therefore, may be advantageouslyemployed for gene silencing via, for example, the RNA interference(RNAi) pathway. That is, partially duplexed dsRNA molecules describedherein (also referred to as meroduplexes or meromers having a nick orgap in at least one strand) are capable of initiating an RNAi cascadethat modifies (e.g., reduces) expression of a target messenger RNA(mRNA) or a family of related target mRNAs. The gene silencingfunctionality of such a structure was unpredictable since thethermodynamically less stable nicked or gapped dsRNA passenger strand(as compared to an intact dsRNA) would be expected to fall apart beforeany gene silencing occurred (see, e.g., Leuschner et al., EMBO 7:314,2006; Bramsen et al., Nucleic Acids Res. 35:5886, 2007).

Meroduplex ribonucleic acid (mdRNA) molecules described herein include afirst (antisense) strand that is complementary to a target mRNA, alongwith second and third strands (together forming a gapped sense strand)that are each complementary to non-overlapping regions of the firststrand, wherein the second and third strands can anneal with the firststrand to form at least two double-stranded regions separated by a gap,and wherein at least one double-stranded region is from about 5 basepairs to 15 base pairs, or the combined double-stranded regions totalabout 15 base pairs to about 40 base pairs and the mdRNA is blunt-ended.

The gap can be from zero nucleotides (i.e., a nick in which only aphosphodiester bond between two nucleotides is broken in apolynucleotide molecule) up to about 10 nucleotides (i.e., the firststrand will have at least non-terminal unpaired nucleotide). In certainembodiments, the nick or gap is located about 10 nucleotides from the5′-end of the first (antisense) strand or at the Argonaute cleavagesite. In another embodiment, the meroduplex nick or gap is positionedsuch that the thermal stability is maximized for the first and secondstrand duplex and for the first and third strand duplex as compared tothe thermal stability of such meroduplexes having a nick or gap in adifferent position.

Also provided herein are methods of using such dsRNA to reduceexpression of a target gene, or one or more genes that are a part of thetarget gene family, in a cell or to treat or prevent diseases ordisorders associated with expression of one or more target gene familymembers, such as hyperproliferative disorders (e.g., cancer),inflammatory conditions (e.g., arthritis), respiratory disease,pulmonary disease, cardiovascular disease, autoimmune disease, allergicdisorders, neurologic disease, infectious disease (e.g., viralinfection, such as influenza), renal disease, transplant rejection, orany other disease or condition that responds to modulation of a targetgene or gene family.

Prior to introducing more detail into this disclosure, it may be helpfulto an appreciation thereof to provide definitions of certain terms to beused herein.

In the present description, any concentration range, percentage range,ratio range, or integer range is to be understood to include the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. Also, any number range recited herein relating toany physical feature, such as polymer subunits, size or thickness, areto be understood to include any integer within the recited range, unlessotherwise indicated. As used herein, “about” or “consisting essentiallyof” mean±20% of the indicated range, value, or structure, unlessotherwise indicated. As used herein, the terms “include” and “comprise”are open-ended and are used synonymously. It should be understood thatthe terms “a” and “an” as used herein refer to “one or more” of theenumerated components. The use of the alternative (e.g., “or”) should beunderstood to mean either one, both, or any combination thereof of thealternatives.

As used herein, “complementary” refers to a nucleic acid molecule thatcan form hydrogen bond(s) with another nucleic acid molecule or itselfby either traditional Watson-Crick base pairing or other non-traditionaltypes of pairing (e.g., Hoogsteen or reversed Hoogsteen hydrogenbonding) between complementary nucleosides or nucleotides. In referenceto the nucleic molecules of the present disclosure, the binding freeenergy for a nucleic acid molecule with its complementary sequence issufficient to allow the relevant function of the nucleic acid moleculeto proceed, for example, RNAi activity, and there is a sufficient degreeof complementarity to avoid non-specific binding of the nucleic acidmolecule (e.g., dsRNA) to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment, or under conditions inwhich the assays are performed in the case of in vitro assays (e.g.,hybridization assays). Determination of binding free energies fornucleic acid molecules is well known in the art (see, e.g., Turner etal., CSH Symp. Quant. Biol. LII:123, 1987; Frier et al., Proc. Nat.Acad. Sci. USA 83:9373, 1986; Turner et al., J. Am. Chem. Soc. 109:3783,1987). Thus, “complementary” or “specifically hybridizable” or“specifically binds” are terms that indicate a sufficient degree ofcomplementarity or precise pairing such that stable and specific bindingoccurs between a nucleic acid molecule (e.g., dsRNA) and a DNA or RNAtarget. It is understood in the art that a nucleic acid molecule neednot be 100% complementary to a target nucleic acid sequence to bespecifically hybridizable or to specifically bind. That is, two or morenucleic acid molecules may be less than fully complementary and isindicated by a percentage of contiguous residues in a nucleic acidmolecule that can form hydrogen bonds with a second nucleic acidmolecule.

For example, a first nucleic acid molecule may have 10 nucleotides and asecond nucleic acid molecule may have 10 nucleotides, then base pairingof 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleicacid molecules, which may or may not form a contiguous double-strandedregion, represents 50%, 60%, 70%, 80%, 90%, and 100% complementarity,respectively. In certain embodiments, complementary nucleic acidmolecules may have wrongly paired bases—that is, bases that cannot forma traditional Watson-Crick base pair or other non-traditional types ofpair (i.e., “mismatched” bases). For instance, fully complementarynucleic acid molecules may be identified as having a certain number of“mismatches,” such as zero or about 1, about 2, about 3, about 4 orabout 5.

“Perfectly” or “fully” complementary nucleic acid molecules means thosein which a certain number of nucleotides of a first nucleic acidmolecule hydrogen bond (anneal) with the same number of residues in asecond nucleic acid molecule to form a contiguous double-strandedregion. For example, two or more fully complementary nucleic acidmolecule strands can have the same number of nucleotides (i.e., have thesame length and form one double-stranded region, with or without anoverhang) or have a different number of nucleotides (e.g., one strandmay be shorter than but fully contained within a second strand or onestrand may overhang the second strand).

As used herein, “ribonucleic acid” or “RNA” means a nucleic acidmolecule comprising at least one ribonucleotide molecule. It should beunderstood that “ribonucleotide” refers to a nucleotide with a hydroxylgroup at the 2′-position of a β-D-ribofuranose moiety. The term RNAincludes double-stranded (ds) RNA, single-stranded (ss) RNA, isolatedRNA (such as partially purified RNA, essentially pure RNA, syntheticRNA, recombinantly produced RNA), altered RNA (which differs fromnaturally occurring RNA by the addition, deletion, substitution oralteration of one or more nucleotides), or any combination thereof. Forexample, such altered RNA can include addition of non-nucleotidematerial, such as at one or both ends of an RNA molecule, internally atone or more nucleotides of the RNA, or any combination thereof.Nucleotides in RNA molecules of the instant disclosure can also comprisenon-standard nucleotides, such as naturally occurring nucleotides,non-naturally occurring nucleotides, chemically-modified nucleotides,deoxynucleotides, or any combination thereof. These altered RNAs may bereferred to as analogs or analogs of RNA containing standard nucleotides(i.e., standard nucleotides, as used herein, are considered to beadenine, cytidine, guanidine, thymidine, and uridine).

The term “dsRNA” as used herein, which is interchangeable with “mdRNA,”refers to any nucleic acid molecule comprising at least oneribonucleotide and is capable of inhibiting or down regulating geneexpression, for example, by promoting RNA interference (“RNAi”) or genesilencing in a sequence-specific manner. The dsRNAs (mdRNAs) of theinstant disclosure may be suitable substrates for Dicer or forassociation with RISC to mediate gene silencing by RNAi. One or bothstrands of the dsRNA can further comprise a terminal phosphate group,such as a 5′-phosphate or 5′,3′-diphosphate. As used herein, dsRNAmolecules, in addition to at least one ribonucleotide, can furtherinclude substitutions, chemically-modified nucleotides, andnon-nucleotides. In certain embodiments, dsRNA molecules compriseribonucleotides up to about 100% of the nucleotide positions.

In addition, as used herein, the term dsRNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example, meroduplex RNA (mdRNA),nicked dsRNA (ndsRNA), gapped dsRNA (gdsRNA), short interfering nucleicacid (siNA), siRNA, micro-RNA (miRNA), short hairpin RNA (shRNA), shortinterfering oligonucleotide, short interfering substitutedoligonucleotide, short interfering modified oligonucleotide,chemically-modified dsRNA, post-transcriptional gene silencing RNA(ptgsRNA), or the like. The term “large double-stranded (ds) RNA” refersto any double-stranded RNA longer than about 40 base pairs (bp) to about100 bp or more, particularly up to about 300 bp to about 500 bp. Thesequence of a large dsRNA may represent a segment of an mRNA or anentire mRNA. A double-stranded structure may be formed byself-complementary nucleic acid molecule or by annealing of two or moredistinct complementary nucleic acid molecule strands.

In one aspect, a dsRNA comprises two separate oligonucleotides,comprising a first strand (antisense) and a second strand (sense),wherein the antisense and sense strands are self-complementary (i.e.,each strand comprises a nucleotide sequence that is complementary to anucleotide sequence in the other strand and the two separate strandsform a duplex or double-stranded structure, for example, wherein thedouble-stranded region is about 15 to about 24 or 25 base pairs or about25 or 26 to about 40 base pairs); the antisense strand comprises anucleotide sequence that is complementary to a nucleotide sequence in atarget nucleic acid molecule or a portion thereof; and the sense strandcomprises a nucleotide sequence corresponding (i.e., homologous) to thetarget nucleic acid sequence or a portion thereof (e.g., a sense strandof about 15 to about 25 nucleotides or about 26 to about 40 nucleotidescorresponds to the target nucleic acid or a portion thereof).

In another aspect, the dsRNA is assembled from a single oligonucleotidein which the self-complementary sense and antisense strands of the dsRNAare linked by together by a nucleic acid based-linker or a non-nucleicacid-based linker. In certain embodiments, the first (antisense) andsecond (sense) strands of the dsRNA molecule are covalently linked by anucleotide or non-nucleotide linker as described herein and known in theart. In other embodiments, a first dsRNA molecule is covalently linkedto at least one second dsRNA molecule by a nucleotide or non-nucleotidelinker known in the art, wherein the first dsRNA molecule can be linkedto a plurality of other dsRNA molecules that can be the same ordifferent, or any combination thereof. In another embodiment, the linkeddsRNA may include a third strand that forms a meroduplex with the linkeddsRNA.

In still another aspect, dsRNA molecules described herein form ameroduplex RNA (mdRNA) having three or more strands such as, forexample, an ‘A’ (first or antisense) strand, ‘S1’ (second) strand, and‘S2’ (third) strand in which the ‘S1’ and ‘S2’ strands are complementaryto and form base pairs (bp) with non-overlapping regions of the ‘A’strand (e.g., an mdRNA can have the form of A:S1S2). The S1, S2, or morestrands together essentially comprise a sense strand to the ‘A’ strand.The double-stranded region formed by the annealing of the ‘S1’ and ‘A’strands is distinct from and non-overlapping with the double-strandedregion formed by the annealing of the ‘S2’ and ‘A’ strands. An mdRNAmolecule is a “gapped” molecule, meaning a “gap” ranging from 0nucleotides up to about 10 nucleotides. In one embodiment, the A:S1duplex is separated from the A:S2 duplex by a gap resulting from atleast one unpaired nucleotide (up to about 10 unpaired nucleotides) inthe ‘A’ strand that is positioned between the A:S1 duplex and the A:S2duplex and that is distinct from any one or more unpaired nucleotide atthe 3′-end of one or more of the ‘A’, ‘S1’, or ‘S2’ strands. In anotherembodiment, the A:S1 duplex is separated from the A:S2 duplex by a gapof zero nucleotides (i.e., a nick in which only a phosphodiester bondbetween two nucleotides is broken or missing in the polynucleotidemolecule) between the A:S1 duplex and the A:S2 duplex—which can also bereferred to as nicked dsRNA (ndsRNA). For example, A:S1S2 may becomprised of a dsRNA having at least two double-stranded regions thatcombined total about 14 base pairs to about 40 base pairs and thedouble-stranded regions are separated by a gap of about 0 to about 10nucleotides, optionally having blunt ends, or A:S1S2 may comprise adsRNA having at least two double-stranded regions separated by a gap ofup to 10 nucleotides wherein at least one of the double-stranded regionscomprises between about 5 base pairs and 13 base pairs.

A dsRNA or large dsRNA may include a substitution or modification inwhich the substitution or modification may be in a phosphate backbonebond, a sugar, a base, or a nucleoside. Such nucleoside substitutionscan include natural non-standard nucleosides (e.g., 5-methyluridine or5-methylcytidine or a 2-thioribothymidine), and such backbone, sugar, ornucleoside modifications can include an alkyl or heteroatom substitutionor addition, such as a methyl, alkoxyalkyl, halogen, nitrogen or sulfur,or other modifications known in the art.

In certain embodiments, the dsRNA or mdRNA will be isolated. As usedherein, the term “isolated” means that the molecule referred to isremoved from its original environment, such as being separated from someor all of the co-existing materials in a natural environment (e.g., anatural environment may be a cell).

As used herein, the term “RNAi” is meant to be equivalent to other termsused to describe sequence specific RNA interference, such as posttranscriptional gene silencing, translational inhibition, orepigenetics. For example, dsRNA molecules of this disclosure can be usedto epigenetically silence genes at the post-transcriptional level or thepre-transcriptional level or any combination thereof.

As used herein, “target nucleic acid” refers to any nucleic acidsequence whose expression or activity is to be altered. The targetnucleic acid can be DNA, RNA, or analogs thereof, and includes single,double, and multi-stranded forms. By “target site” or “target sequence”is meant a sequence within a target nucleic acid (e.g., mRNA) that is“targeted” for cleavage by RNAi and mediated by a dsRNA construct ofthis disclosure containing a sequence within the antisense strand thatis complementary to the target site or sequence.

As used herein, “off-target effect” or “off-target profile” refers tothe observed altered expression pattern of one or more genes in a cellor other biological sample not targeted, directly or indirectly, forgene silencing by an mdRNA or dsRNA. For example, an off-target effectcan be quantified by using a DNA microarray to determine how manynon-target genes have an expression level altered by about 2-fold ormore in the presence of a candidate mdRNA or dsRNA, or analog thereofspecific for a target sequence, such as one or more target mRNA. A“minimal off-target effect” means that an mdRNA or dsRNA affectsexpression by about 2-fold or more of about 25% to about 1% of thenon-target genes examined or it means that the off-target effect ofsubstituted or modified mdRNA or dsRNA (e.g., having at least oneuridine substituted with a 5-methyluridine or 2-thioribothymidine andoptionally having at least one nucleotide modified at the 2′-position),is reduced by at least about 1% to about 80% or more as compared to theeffect on non-target genes of an unsubstituted or unmodified mdRNA ordsRNA.

By “sense region” or “sense strand” is meant one or more nucleotidesequences of a dsRNA molecule having complementarity to one or moreantisense regions of the dsRNA molecule. In addition, the sense regionof a dsRNA molecule comprises a nucleic acid sequence having homology oridentity to a target sequence. By “antisense region” or “antisensestrand” is meant a nucleotide sequence of a dsRNA molecule havingcomplementarity to a target nucleic acid sequence. In addition, theantisense region of a dsRNA molecule can comprise a nucleic acidsequence regions having complementarity to one or more sense strands ofa dsRNA molecule.

“Analog” as used herein refers to a compound that is structurallysimilar to a parent compound (e.g., a nucleic acid molecule), butdiffers slightly in composition (e.g., one atom or functional group isdifferent, added, or removed). The analog may or may not have differentchemical or physical properties than the original compound and may ormay not have improved biological or chemical activity. For example, theanalog may be more hydrophilic or it may have altered activity ascompared to a parent compound. The analog may mimic the chemical orbiological activity of the parent compound (i.e., it may have similar oridentical activity), or, in some cases, may have increased or decreasedactivity. The analog may be a naturally or non-naturally occurring(e.g., chemically-modified or recombinant) variant of the originalcompound. An example of an RNA analog is an RNA molecule having anon-standard nucleotide, such as 5-methyuridine or 5-methylcytidine or2-thioribothymidine, which may impart certain desirable properties(e.g., improve stability, bioavailability, minimize off-target effectsor interferon response).

The term “pyrimidine” as used herein refers to conventional pyrimidinebases, including standard pyrimidine bases uracil and cytosine. Inaddition, the term pyrimidine is contemplated to embrace naturalnon-standard pyrimidine bases or acids, such as 5-methyluracil,2-thio-5-methyluracil, 4-thiouracil, pseudouracil, dihydrouracil,orotate, 5-methylcytosine, or the like, as well as a chemically-modifiedbases or “universal bases,” which can be used to substitute for astandard pyrimidine within nucleic acid molecules of this disclosure.

The term “purine” as used herein refers to conventional purine bases,including standard purine bases adenine and guanine. In addition, theterm purine is contemplated to embrace natural non-standard purine basesor acids, such as N2-methylguanine, inosine, or the like, as well as achemically-modified bases or “universal bases,” which can be used tosubstitute for a standard purine within nucleic acid molecules of thisdisclosure.

As used herein, the term “universal base” refers to nucleotide baseanalogs that form base pairs with each of the standard DNA/RNA baseswith little discrimination between them, and is recognized byintracellular enzymes (see, e.g., Loakes et al., J. Mol. Bio.270:426-435, 1997). Non-limiting examples of universal bases includeC-phenyl, C-naphthyl and other aromatic derivatives, inosine, azolecarboxamides, and nitroazole derivatives such as 3-nitropyrrole,4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art(see, e.g., Loakes, Nucleic Acids Res. 29:2437-2447, 2001).

The term “gene” as used herein, especially in the context of “targetgene” or “gene target” for RNAi, means a nucleic acid molecule thatencodes an RNA or a transcription product of such gene, including amessenger RNA (mRNA, also referred to as structural genes that encodefor a polypeptide), an mRNA splice variant of such gene, a functionalRNA (fRNA), or non-coding RNA (ncRNA), such as small temporal RNA(stRNA), microRNA (miRNA), small nuclear RNA (snRNA), short interferingRNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transferRNA (tRNA) and precursor RNAs thereof. Such non-coding RNAs can serve astarget nucleic acid molecules for dsRNA mediated RNAi to alter theactivity of fRNA or ncRNA involved in functional or regulatory cellularprocesses. A target gene can be a gene derived from a cell, such as anendogenous gene, a transgene, or exogenous gene, including genes from apathogen (e.g., a viral gene) that is present in a cell after infectionthereof. A cell containing a target gene can be derived from orcontained in any organism, for example, a plant, animal, protozoan,virus, bacterium, or fungus.

Furthermore, one or more dsRNA may be used to knockdown expression of atarget mRNA or a related mRNA splice variant. In this regard, it isnoted that a target gene may be transcribed into two or more mRNA splicevariants. In certain embodiments, knockdown of one target mRNA splicevariant without affecting one or more other target mRNA splice variantsmay be desired, or vice versa. Alternatively, knockdown of alltranscription products of one or more target family genes iscontemplated herein.

As used herein, “gene silencing” refers to a partial or completeloss-of-function through targeted inhibition of gene expression in acell, which may also be referred to as RNAi “knockdown,” “inhibition,”“down-regulation,” or “reduction” of expression of a target gene.Depending on the circumstances and the biological problem to beaddressed, it may be preferable to partially reduce gene expression.Alternatively, it might be desirable to reduce gene expression as muchas possible. The extent of silencing may be determined by methodsdescribed herein and known in the art, some of which are summarized inPCT Publication No. WO 99/32619. Depending on the assay, quantificationof gene expression permits detection of various amounts of inhibitionthat may be desired in certain embodiments of this disclosure, includingprophylactic and therapeutic methods, which will be capable of knockingdown target gene expression, in terms of mRNA level or protein level oractivity, for example, by equal to or greater than 10%, 30%, 50%, 75%90%, 95% or 99% of baseline (i.e., normal) or other control levels,including elevated expression levels as may be associated withparticular disease states or other conditions targeted for therapy.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of this disclosure can beadministered. In one embodiment, a subject is a mammal or mammaliancell. In another embodiment, a subject is a human or human cell.

As used herein, the term “therapeutically effective amount” means anamount of dsRNA that is sufficient to result in a decrease in severityof disease symptoms, an increase in frequency or duration of diseasesymptom-free periods, or a prevention of impairment or disability due tothe disease, in the subject (e.g., mammal or human) to which it isadministered. For example, a therapeutically effective amount of dsRNAdirected against a target mRNA, which effectively down-regulates thetarget-encoding mRNA and thereby reduces or prevents one or moretarget-associated disorders, such as an infection, inflammation,metabolic disorders, autoimmune condition(s), cancer, or the like. Oneof ordinary skill in the art would be able to determine suchtherapeutically effective amounts based on such factors as the subject'ssize, the severity of symptoms, and the particular composition or routeof administration selected. For example, a therapeutically effectiveamount of a compound can decrease tumor size or otherwise amelioratesymptoms associated with a particular disorder in a subject. The dsRNAmolecules of the instant disclosure, individually or in combination orin conjunction with other drugs, can be used to treat diseases orconditions discussed herein, by administering to a subject or byadministering to particular cells under conditions suitable fortreatment.

In addition, it should be understood that the individual compounds, orgroups of compounds, derived from the various combinations of thestructures and substituents described herein, are disclosed by thepresent application to the same extent as if each compound or group ofcompounds was set forth individually. Thus, selection of particularstructures or particular substituents is within the scope of the presentdisclosure. As described herein, all value ranges are inclusive over theindicated range. Thus, a range of C₁-C₄ will be understood to includethe values of 1, 2, 3, and 4, such that C₁, C₂, C₃ and C₄ are included.

The term “alkyl” as used herein refers to saturated straight- orbranched-chain aliphatic groups containing from 1-20 carbon atoms,preferably 1-8 carbon atoms and most preferably 1-4 carbon atoms. Thisdefinition applies as well to the alkyl portion of alkoxy, alkanoyl andaralkyl groups. The alkyl group may be substituted or unsubstituted. Incertain embodiments, the alkyl is a (C₁-C₄) alkyl or methyl.

The term “cycloalkyl” as used herein refers to a saturated cyclichydrocarbon ring system containing from 3 to 12 carbon atoms that may beoptionally substituted. Exemplary embodiments include, but are notlimited to, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Incertain embodiments, the cycloalkyl group is cyclopropyl. In anotherembodiment, the (cycloalkyl)alkyl groups contain from 3 to 12 carbonatoms in the cyclic portion and 1 to 6 carbon atoms in the alkylportion. In certain embodiments, the (cycloalkyl)alkyl group iscyclopropylmethyl. The alkyl groups are optionally substituted with fromone to three substituents selected from the group consisting of halogen,hydroxy and amino.

The terms “alkanoyl” and “alkanoyloxy” as used herein refer,respectively, to —C(O)-alkyl groups and —O—C(═O)— alkyl groups, eachoptionally containing 2 to 10 carbon atoms. Specific embodiments ofalkanoyl and alkanoyloxy groups are acetyl and acetoxy, respectively.

The term “alkenyl” refers to an unsaturated branched, straight-chain orcyclic alkyl group having 2 to 15 carbon atoms and having at least onecarbon-carbon double bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkene. The group may be in eitherthe cis or trans conformation about the double bond(s). Certainembodiments include ethenyl, 1-propenyl, 2-propenyl, 1-methylethenyl,1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-2-propenyl, 1-pentenyl,2-pentenyl, 4-pentenyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl,1-heptenyl, 2-heptenyl, 1-octenyl, 2-octenyl, 1,3-octadienyl, 2-nonenyl,1,3-nonadienyl, 2-decenyl, etc., or the like. The alkenyl group may besubstituted or unsubstituted.

The term “alkynyl” as used herein refers to an unsaturated branched,straight-chain, or cyclic alkyl group having 2 to 10 carbon atoms andhaving at least one carbon-carbon triple bond derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkyne.Exemplary alkynyls include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 4-pentynyl, 1-octynyl,6-methyl-1-heptynyl, 2-decynyl, or the like. The alkynyl group may besubstituted or unsubstituted.

The term “hydroxyalkyl” alone or in combination, refers to an alkylgroup as previously defined, wherein one or several hydrogen atoms,preferably one hydrogen atom has been replaced by a hydroxyl group.Examples include hydroxymethyl, hydroxyethyl and 2-hydroxyethyl.

The term “aminoalkyl” as used herein refers to the group —NRR′, where Rand R′ may independently be hydrogen or (C₁-C₄) alkyl.

The term “alkylaminoalkyl” refers to an alkylamino group linked via analkyl group (i.e., a group having the general structure -alkyl-NH-alkylor -alkyl-N(alkyl)(alkyl)). Such groups include, but are not limited to,mono- and di-(C₁-C₈ alkyl)aminoC₁-C₈ alkyl, in which each alkyl may bethe same or different.

The term “dialkylaminoalkyl” refers to alkylamino groups attached to analkyl group. Examples include, but are not limited to,N,N-dimethylaminomethyl, N,N-dimethylaminoethyl N,N-dimethylaminopropyl,and the like. The term dialkylaminoalkyl also includes groups where thebridging alkyl moiety is optionally substituted.

The term “haloalkyl” refers to an alkyl group substituted with one ormore halo groups, for example, chloromethyl, 2-bromoethyl, 3-iodopropyl,trifluoromethyl, perfluoropropyl, 8-chlorononyl, or the like.

The term “carboxyalkyl” as used herein refers to the substituent—R^(Z)—COOH, wherein R¹⁰ is alkylene; and carbalkoxyalkyl refers to—R¹⁰—C(O)OR¹¹, wherein R¹⁰ and R¹¹ are alkylene and alkyl respectively.In certain embodiments, alkyl refers to a saturated straight- orbranched-chain hydrocarbyl radical of 1 to 6 carbon atoms such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl,2-methylpentyl, n-hexyl, and so forth. Alkylene is the same as alkylexcept that the group is divalent.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. In oneembodiment, the alkoxy group contains 1 to about 10 carbon atoms.Embodiments of alkoxy groups include, but are not limited to, methoxy,ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Embodimentsof substituted alkoxy groups include halogenated alkoxy groups. In afurther embodiment, the alkoxy groups can be substituted with groupssuch as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Exemplary halogen substituted alkoxy groupsinclude, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy, and trichloromethoxy.

The term “alkoxyalkyl” refers to an alkylene group substituted with analkoxy group. For example, methoxyethyl (CH₃OCH₂CH₂—) and ethoxymethyl(CH₃CH₂OCH₂—) are both C₃ alkoxyalkyl groups.

The term “aryl” as used herein refers to monocyclic or bicyclic aromatichydrocarbon groups having from 6 to 12 carbon atoms in the ring portion,for example, phenyl, naphthyl, biphenyl and diphenyl groups, each ofwhich may be substituted with, for example, one to four substituentssuch as alkyl; substituted alkyl as defined above, halogen,trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, cycloalkyloxy,alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino, nitro, cyano,carboxy, carboxyalkyl, carbamyl, carbamoyl and aryloxy. Specificembodiments of aryl groups in accordance with the present disclosureinclude phenyl, substituted phenyl, naphthyl, biphenyl, and diphenyl.

The term “aroyl” as used alone or in combination herein refers to anaryl radical derived from an aromatic carboxylic acid, such asoptionally substituted benzoic or naphthoic acids.

The term “aralkyl” as used herein refers to an aryl group bonded to the2-pyridinyl ring or the 4-pyridinyl ring through an alkyl group,preferably one containing 1 to 10 carbon atoms. A preferred aralkylgroup is benzyl.

The term “carboxy” as used herein represents a group of the formula—C(═O)OH or —C(═O)O⁻.

The term “carbonyl” as used herein refers to a group in which an oxygenatom is double-bonded to a carbon atom —C═O.

The term “trifluoromethyl” as used herein refers to —CF₃.

The term “trifluoromethoxy” as used herein refers to —OCF₃.

The term “hydroxyl” as used herein refers to —OH or —O⁻.

The term “nitrile” or “cyano” as used herein refers to the group —CN.

The term “nitro” as used herein alone or in combination refers to a —NO₂group.

The term “amino” as used herein refers to the group —NR⁹R⁹, wherein R⁹may independently be hydrogen, alkyl, aryl, alkoxy, or heteroaryl. Theterm “aminoalkyl” as used herein represents a more detailed selection ascompared to “amino” and refers to the group —NR′R′, wherein R′ mayindependently be hydrogen or (C₁-C₄) alkyl. The term “dialkylamino”refers to an amino group having two attached alkyl groups that can bethe same or different.

The term “alkanoylamino” refers to alkyl, alkenyl or alkynyl groupscontaining the group —C(═O)— followed by —N(H)—, for example,acetylamino, propanoylamino and butanoylamino and the like.

The term “carbonylamino” refers to the group —NR′—CO—CH₂—R′, wherein R′is independently selected from hydrogen or (C₁-C₄) alkyl.

The term “carbamoyl” as used herein refers to —O—C(O)NH₂.

The term “carbamyl” as used herein refers to a functional group in whicha nitrogen atom is directly bonded to a carbonyl, i.e., as in—NR′C(═O)R′ or —C(═O)NR′R′, wherein R′ can be independently hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,cycloalkyl, aryl, heterocyclo, or heteroaryl.

The term “alkylsulfonylamino” refers to refers to the group —NHS(O)₂R¹²,wherein R¹² is alkyl.

The term “halogen” as used herein refers to bromine, chlorine, fluorineor iodine. In one embodiment, the halogen is fluorine. In anotherembodiment, the halogen is chlorine.

The term “heterocyclo” refers to an optionally substituted, unsaturated,partially saturated, or fully saturated, aromatic or nonaromatic cyclicgroup that is a 4 to 7 membered monocyclic, or 7 to 11 membered bicyclicring system that has at least one heteroatom in at least one carbonatom-containing ring. The substituents on the heterocyclo rings may beselected from those given above for the aryl groups. Each ring of theheterocyclo group containing a heteroatom may have 1, 2, or 3heteroatoms selected from nitrogen, oxygen or sulfur. Plural heteroatomsin a given heterocyclo ring may be the same or different.

Exemplary monocyclic heterocyclo groups include pyrrolidinyl, pyrrolyl,indolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl,tetrahydrofuryl, thienyl, piperidinyl, piperazinyl, azepinyl,pyrimidinyl, pyridazinyl, tetrahydropyranyl, morpholinyl, dioxanyl,triazinyl and triazolyl. Preferred bicyclic heterocyclo groups includebenzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl,tetrahydroisoquinolinyl, benzimidazolyl, benzofuryl, indazolyl,benzisothiazolyl, isoindolinyl and tetrahydroquinolinyl. In moredetailed embodiments heterocyclo groups may include indolyl, imidazolyl,furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl and pyrimidyl.

“Substituted” refers to a group in which one or more hydrogen atoms areeach independently replaced with the same or different substituent(s).Representative substituents include —X, —R⁶, —O—, ═O, —OR, —SR⁶, —S—,═S, —NR⁶R⁶, ═NR⁶, —CX₃, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃,—S(═O)₂O—, —S(═O)₂OH, —S(═O)₂R⁶, —OS(═O)₂O—, —OS(═O)₂OH, —OS(═O)₂R⁶,—P(═O)(O⁻)₂, —P(═O)(OH)(O⁻), —OP(═O)₂(O), —C(—O)R⁶, —C(═S)R⁶, —C(═O)OR⁶,—C(═O)O⁻, —C(═S)OR⁶, —NR⁶—C(═O)—N(R⁶)₂, —NR⁶—C(═S)—N(R⁶)₂, and—C(═NR⁶)NR⁶R⁶, wherein each X is independently a halogen; and each R⁶ isindependently hydrogen, halogen, alkyl, aryl, arylalkyl, arylaryl,arylheteroalkyl, heteroaryl, heteroarylalkyl, NR⁷R⁷, —C(═O)R⁷, and—S(═O)₂R⁷; and each R⁷ is independently hydrogen, alkyl, alkanyl,alkynyl, aryl, arylalkyl, arylheteralkyl, arylaryl, heteroaryl orheteroarylalkyl. Aryl containing substituents, whether or not having oneor more substitutions, may be attached in a para (p-), meta (m-) orortho (o-) conformation, or any combination thereof.

Gapped or Nicked dsRNA Molecules

This disclosure provides compounds, compositions, and methods useful foraltering expression or activity of a target gene by RNA interference(RNAi) using small nucleic acid molecules. In more detailed embodiments,this disclosure provides small nucleic acid molecules, such as shortinterfering nucleic acid (siNA), short interfering RNA (siRNA),double-stranded RNA (dsRNA), nicked double-stranded RNA (ndsRNA), gappeddouble-stranded RNA (gdsRNA), microRNA (miRNA), short hairpin RNA(shRNA) molecules, or any combination thereof, which have a at least onenick or gap and alter expression of a target gene or family of genes toprevent, treat, or alleviate symptoms of a disease or disorder in asubject (e.g., human). Within these and related therapeutic compositionsand methods, the use of nicked or gapped dsRNAs (which have beenoptionally substituted or modified) will often improve properties of thedsRNA molecules in comparison to the properties of native dsRNAmolecules, such as reduced off-target effects, reduced interferonresponse, increased resistance to nuclease degradation in vivo, improvedcellular uptake, increased potency, or any combination thereof.

In particular embodiments, there are provided methods of treating orpreventing diseases, disorders, or conditions related to geneexpression, including those related, or responsive, to the level of atarget nucleic acid molecule (e.g., mRNA) in a cell or tissue, byadministering a gapped dsRNA (mdRNA) molecule of this disclosure, aloneor in combination with an adjunctive therapy, in an amount sufficient toactivate target gene-specific RNAi. In one embodiment, there is provideda method of treating or preventing a disease or disorder byadministering a dsRNA molecule that is capable of target gene-specificRNAi, which dsRNA has at least one substitution or modification asdescribed herein and has a reduced or minimal off-target effect.

The “percent identity” between two or more nucleic acid sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity=number of identical positions/total number ofpositions×100), taking into account the number of gaps, and the lengthof each gap that needs to be introduced to optimize alignment of two ormore sequences. The comparison of sequences and determination of percentidentity between two or more sequences can be accomplished using amathematical algorithm, such as BLAST and Gapped BLAST programs at theirdefault parameters (e.g., Altschul et al., J. Mol. Biol. 215:403, 1990;see also BLASTN at www.ncbi.nlm.nih.gov/BLAST).

In one aspect, the instant disclosure provides a meroduplex ribonucleicacid (mdRNA) molecule, comprising a first strand that is complementaryto a target mRNA, and a second strand and a third strand that is eachcomplementary to non-overlapping regions of the first strand, whereinthe second strand and third strands can anneal with the first strand toform at least two double-stranded regions separated by a gap of up to 10nucleotides, and wherein (a) at least one double-stranded regioncomprises from about 5 base pairs to 13 base pairs, or (b) wherein thecombined double-stranded regions total about 15 base pairs to about 40base pairs and the mdRNA molecule comprises blunt ends; wherein at leastone pyrimidine of the mdRNA is substituted with a pyrimidine nucleosideaccording to Formula I or II:

wherein R¹ and R² are each independently a —H, —OH, —OCH₃, —OCH₂OCH₂CH₃,—OCH₂CH₂OCH₃, halogen, substituted or unsubstituted C₁-C₁₀ alkyl,alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino,aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl,trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted —O-allyl,—O—CH₂CH═CH₂, —O—CH═CHCH₃, substituted or unsubstituted C₂-C₁₀ alkynyl,carbamoyl, carbamyl, carboxy, carbonylamino, substituted orunsubstituted aryl, substituted or unsubstituted aralkyl, —NH₂, —NO₂, orheterocyclo group; R³ and R⁴ are each independently a hydroxyl, aprotected hydroxyl, a phosphate, or an internucleoside linking group;and R⁵ and R⁸ are independently O or S. In certain embodiments, at leastone nucleoside is according to Formula I in which R¹ is methyl and R² is—OH, or R¹ is methyl, R² is —OH and R⁸ is S.

In other embodiments, the internucleoside linking group covalently linksfrom about 5 to about 40 nucleosides. In some embodiments, the gapcomprises at least one unpaired nucleotide in the first strandpositioned between the double-stranded regions formed by the second andthird strands when annealed to the first strand, or the gap comprises anick. In certain embodiments, the nick or gap is located about 10nucleotides from the 5′-end of the first strand or at the Argonautecleavage site. In another embodiment, the meroduplex nick or gap ispositioned such that the thermal stability is maximized for the firstand second strand duplex and for the first and third strand duplex ascompared to the thermal stability of such meroduplexes having a nick orgap in a different position—that is, the nick or gap is located in aposition wherein each of the two or more nicked or gapped strands has amaximal melting temperature when annealed to the first strand (i.e.,T_(m) or temperature at which 50% of one of the nicked or gapped strandsis annealed to the first strand).

As provided herein, any of the aspects or embodiments disclosed hereinwould be useful in treating target gene associated diseases ordisorders, such as hyperproliferative disease (e.g., cervical cancer,ovarian cancer), angiogenic disorders (e.g., tumor angiogenesis), orinflammatory disorders (e.g. rheumatoid arthritis, rheumatoid arthritis,chronic obstructive bowel disease, atherosclerosis), respiratorydisease, pulmonary disease, cardiovascular disease, autoimmune disease,allergic disorders, neurologic disease, infectious disease (e.g., viralinfection, such as influenza), renal disease, transplant rejection, orany other disease or condition that responds to modulation of a targetgene or gene family. In certain embodiments of the instant disclosure, asingle dsRNA can be used to knockdown mRNA expression of one or moretarget gene family member.

In some embodiments, the dsRNA comprises at least three strands in whichthe first strand comprises about 5 nucleotides to about 40 nucleotides,and the second and third strands include each, individually, about 5nucleotides to about 20 nucleotides, wherein the combined length of thesecond and third strands is about 15 nucleotides to about 40nucleotides. In other embodiments, the dsRNA comprises at least two orthree strands in which the first strand comprises about 15 nucleotidesto about 24 nucleotides or about 25 nucleotides to about 40 nucleotides.In further embodiments, the first strand will be complementary to asecond strand or a second and third strand or to a plurality of strands.In further examples, the first strand and its complement(s) will be ableto form dsRNA or mdRNA molecules of this disclosure with about 19 toabout 25 nucleotides of the first strand that is complementary to atleast one target gene mRNA.

For example, a Dicer substrate dsRNA can have about 25 nucleotides toabout 40 nucleotides with only 19 nucleotides of the antisense (first)strand being complementary to at least one target gene family mRNA. Infurther embodiments, the first strand can have complementarity with atarget gene family mRNA in about 19 nucleotides to about 25 nucleotidesand have one, two, or three mismatches, or any combination thereof, witha target gene family mRNA or any combination thereof, or the firststrand of about 19 nucleotides to about 25 nucleotides (that, e.g.,activates or is capable of loading into or associating with RISC) canhave at least 80% identity with the corresponding nucleotides found inat least one target gene family mRNA, or any combination thereof.

Substituted and Modified Nicked or Gapped dsRNA Molecules

The introduction of substituted and modified nucleotides into mdRNA anddsRNA molecules of this disclosure provides a tool for overcomingpotential limitations of in vivo stability and bioavailability inherentto native RNA molecules (i.e., having standard nucleotides) that areexogenously delivered. In certain embodiments, the use of substituted ormodified dsRNA molecules of this disclosure can enable a lower dose of aparticular nucleic acid molecule for a given therapeutic effect since ofdsRNA molecules may be designed to have an increased melting temperatureor half-life in a subject or biological samples (e.g., serum).Furthermore, certain substitutions or modifications can be used toimprove the bioavailability of dsRNA by targeting particular cells ortissues or improving cellular uptake of the dsRNA molecules. Therefore,even if the activity of a dsRNA molecule of this disclosure is reducedas compared to a native RNA molecule, the overall activity of thesubstituted or modified dsRNA molecule can be greater than that of thenative RNA molecule due to improved stability or delivery of themolecule. The mdRNA structure may result in a reduced interferonresponse, and substituted and modified dsRNA can also minimize thepossibility of activating an interferon response in, for example,humans.

In certain embodiments, a dsRNA molecule of this disclosure has at leastone uridine, at least three uridines, or each and every uridine (i.e.,all uridines) of the first (antisense) strand of the dsRNA substitutedor replaced with 5-methyluridine or 2-thioribothymidine. In a relatedembodiment, the dsRNA molecule or analog thereof of this disclosure hasat least one uridine, at least three uridines, or each and every uridineof the second (sense) strand of the dsRNA substituted or replaced with5-methyluridine or 2-thioribothymidine. In still another embodiment, thedsRNA molecule or analog thereof of this disclosure has at least oneuridine, at least three uridines, or each and every uridine of both thefirst (antisense) and second (sense) strands of the dsRNA substituted orreplaced with 5-methyluridine or 2-thioribothymidine. In someembodiments, the double-stranded region of a dsRNA molecule has at leastthree 5-methyluridines or 2-thioribothymidines. In certain embodiments,dsRNA molecules comprise ribonucleotides at about 5% to about 95% of thenucleotide positions in one strand, both strands, or any combinationthereof.

In further embodiments, a dsRNA molecule that decreases expression ofone or more target gene by RNAi according to the instant disclosurefurther comprises one or more natural or synthetic non-standardnucleoside. In related embodiments, the non-standard nucleoside is oneor more deoxyuridine, L- or D-locked nucleic acid (LNA) molecule (e.g.,a 5-methyluridine LNA) or substituted LNA (e.g., having a pyrene), or auniversal-binding nucleotide, or a G clamp, or any combination thereof.In certain embodiments, the universal-binding nucleotide can beC-phenyl, C-naphthyl, inosine, azole carboxamide,1-β-D-ribofuranosyl-4-nitroindole, 1-β-D-ribofuranosyl-5-nitroindole,1-β-D-ribofuranosyl-6-nitroindole, or 1-β-D-ribofuranosyl-Substituted ormodified nucleotides present in dsRNA molecules, preferably in theantisense strand, but also optionally in the sense or both the antisenseand sense strands, comprise modified or substituted nucleotidesaccording to this disclosure having properties or characteristicssimilar to natural or standard ribonucleotides. For example, thisdisclosure features dsRNA molecules including nucleotides having aNorthern conformation (e.g., Northern pseudorotation cycle, see, e.g.,Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed.,1984). As such, chemically modified nucleotides present in dsRNAmolecules of this disclosure, preferably in the antisense strand, butalso optionally in the sense or both the antisense and sense strands,are resistant to nuclease degradation while at the same time maintainingthe capacity to mediate RNAi. Exemplary nucleotides having a Northernconfiguration include locked nucleic acid (LNA) nucleotides (e.g., 2′O,4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-methoxyethyl (MOE)nucleotides, 2′methyl-thio-ethyl, 2′deoxy-2′fluoro nucleotides,2′deoxy-2′chloro nucleotides, 2′azido nucleotides, 5-methyluridines, or2′O-methyl nucleotides. In certain embodiments, the LNA is a5-methyluridine LNA or 2-thioribothymidine LNA. In any of theseembodiments, one or more substituted or modified nucleotides can be a Gclamp (e.g., a cytosine analog that forms an additional hydrogen bond toguanine, such as 9-(aminoethoxy)phenoxazine; see, e.g., Lin andMateucci, J. Am. Chem. Soc. 120:8531, 1998).

As described herein, the first and one or more second strands of a dsRNAmolecule or analog thereof provided by this disclosure can anneal orhybridize together (i.e., due to complementarity between the strands) toform at least one double-stranded region having a length of about 4 toabout 10 base pairs, about 5 to about 13 base pairs, or about 15 toabout 40 base pairs. In some embodiments, the dsRNA has at least onedouble-stranded region ranging in length from about 15 to about 24 basepairs or about 19 to about 23 base pairs. In other embodiments, thedsRNA has at least one double-stranded region ranging in length fromabout 26 to about 40 base pairs or about 27 to about 30 base pairs orabout 30 to about 35 base pairs. In other embodiments, the two or morestrands of a dsRNA molecule of this disclosure may optionally becovalently linked together by nucleotide or non-nucleotide linkermolecules.

In certain embodiments, the dsRNA molecule or analog thereof comprisesan overhang of one to four nucleotides on one or both 3′-ends of thedsRNA, such as an overhang comprising a deoxyribonucleotide or twodeoxyribonucleotides (e.g., thymidine, adenine). In certain embodiments,the 3′-end comprising one or more deoxyribonucleotide is in an mdRNAmolecule and is either in the gap, not in the gap, or any combinationthereof. In some embodiments, dsRNA molecules or analogs thereof have ablunt end at one or both ends of the dsRNA. In certain embodiments, the5′-end of the first or second strand is phosphorylated. In any of theembodiments of dsRNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise ribonucleotides ordeoxyribonucleotides that are chemically-modified at a nucleic acidsugar, base, or backbone. In any of the embodiments of dsRNA moleculesdescribed herein, the 3′-terminal nucleotide overhangs can comprise oneor more universal base ribonucleotides. In any of the embodiments ofdsRNA molecules described herein, the 3′-terminal nucleotide overhangscan comprise one or more acyclic nucleotides. In any of the embodimentsof dsRNA molecules described herein, the dsRNA can further comprise aterminal phosphate group, such as a 5′phosphate (see Martinez et al.,Cell 110:563, 2002; and Schwarz et al., Molec. Cell 10:537, 2002) or a5′3′diphosphate.

As set forth herein, the terminal structure of dsRNAs of this disclosurethat decrease expression of one or more target gene by, for example,RNAi may either have blunt ends or one or more overhangs. In certainembodiments, the overhang may be at the 3′end or the 5′end. The totallength of dsRNAs having overhangs is expressed as the sum of the lengthof the paired double-stranded portion together with the overhangingnucleotides. For example, if a 19 base pair dsRNA has a two nucleotideoverhang at both ends, the total length is expressed as 21-mer.Furthermore, since the overhanging sequence may have low specificity toone or more target gene, it is not necessarily complementary (antisense)or identical (sense) to a target gene sequence. In further embodiments,a dsRNA of this disclosure that decreases expression of one or moretarget gene by RNAi may further comprise a low molecular weightstructure (e.g., a natural RNA molecule such as a tRNA, rRNA or viralRNA, or an artificial RNA molecule) at, for example, one or moreoverhanging portion of the dsRNA.

In further embodiments, a dsRNA molecule that decreases expression ofone or more target gene by RNAi according to the instant disclosurecomprises a 2′-sugar substitution, such as a 2′-deoxy,2′-O-2-methoxyethyl, 2′-O-methoxyethyl, 2′-O-methyl, halogen, 2′-fluoro,2′-O-allyl, or the like, or any combination thereof. In still furtherembodiments, a dsRNA molecule that decreases expression of one or moretarget gene by RNAi according to the instant disclosure furthercomprises a terminal cap substituent on one or both ends of the firststrand or one or more second strands, such as an alkyl, abasic, deoxyabasic, glyceryl, dinucleotide, acyclic nucleotide, inverteddeoxynucleotide moiety, or any combination thereof. In certainembodiments, at least one or two 5′-terminal ribonucleotides of thesense strand within the double-stranded region have a 2′-sugarsubstitution. In certain other embodiments, at least one or two5′-terminal ribonucleotides of the antisense strand within thedouble-stranded region have a 2′-sugar substitution. In certainembodiments, at least one or two 5′-terminal ribonucleotides of thesense strand and the antisense strand within the double-stranded regionhave a 2′-sugar substitution.

In other embodiments, a dsRNA molecule that decreases expression of oneor more target gene by RNAi according to the instant disclosurecomprises one or more substitutions in the sugar backbone, including anycombination of ribosyl, 2′-deoxyribosyl, a tetrofuranosyl (e.g.,L-α-threofuranosyl), a hexopyranosyl (e.g., β-allopyranosyl,β-altropyranosyl, and (3-glucopyranosyl), a pentopyranosyl (e.g.,β-ribopyranosyl, α-lyxopyranosyl, β-xylopyranosyl, andα-arabinopyranosyl), a carbocyclic (carbon only ring) analog, apyranose, a furanose, a morpholino, or analogs or derivatives thereof.

In yet other embodiments, a dsRNA molecule that decreases expression ofone or more target gene by RNAi according to the instant disclosurecomprises at least one modified internucleoside linkage, such asindependently a phosphorothioate, chiral phosphorothioate,phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methylphosphonate, alkyl phosphonate, 3′-alkylene phosphonate, 5′-alkylenephosphonate, chiral phosphonate, phosphonoacetate, thiophosphonoacetate,phosphinate, phosphoramidate, 3′-amino phosphoramidate,aminoalkylphosphoramidate, thionophosphoramidate,thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate,boranophosphate linkage, or any combination thereof.

A modified internucleotide linkage, as described herein, can be presentin one or more strands of a dsRNA molecule of this disclosure, forexample, in the sense strand, the antisense strand, both strands, or aplurality of strands (e.g., in an mdRNA). The dsRNA molecules of thisdisclosure can comprise one or more modified internucleotide linkages atthe 3′end, the 5′end, or both of the 3′ and 5′ends of the sense strandor the antisense strand or both strands. In one embodiment, a dsRNAmolecule capable of decreasing expression of one or more target gene byRNAi has one modified internucleotide linkage at the 3′-end, such as aphosphorothioate linkage. For example, this disclosure provides a dsRNAmolecule capable of decreasing expression of one or more target gene byRNAi having about 1 to about 8 or more phosphorothioate internucleotidelinkages in one dsRNA strand. In yet another embodiment, this disclosureprovides a dsRNA molecule capable of decreasing expression of one ormore target gene by RNAi having about 1 to about 8 or morephosphorothioate internucleotide linkages in both dsRNA strands. Inother embodiments, an exemplary dsRNA molecule of this disclosure cancomprise from about 1 to about 5 or more consecutive phosphorothioateinternucleotide linkages at the 5′end of the sense strand, the antisensestrand, both strands, or a plurality of strands. In another example, anexemplary dsRNA molecule of this disclosure can comprise one or morepyrimidine phosphorothioate internucleotide linkages in the sensestrand, the antisense strand, two strands, or a plurality of strands. Inyet another example, an exemplary dsRNA molecule of this disclosure cancomprise one or more purine phosphorothioate internucleotide linkages inthe sense strand, the antisense strand, two strands, or a plurality ofstrands.

Many exemplary modified nucleotide bases or analogs thereof useful inthe dsRNA of the instant disclosure include 5-methylcytosine;5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine;6-methyl, 2-propyl, or other alkyl derivatives of adenine and guanine;8-substituted adenines and guanines (such as 8-aza, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl, or the like); 7-methyl, 7-deaza, and3-deaza adenines and guanines; 2-thiouracil; 2-thiothymine;2-thiocytosine; 5-methyl, 5-propynyl, 5-halo (such as 5-bromo or5-fluoro), 5-trifluoromethyl, or other 5-substituted uracils andcytosines; and 6-azouracil. Further useful nucleotide bases can be foundin Kurreck, Eur. J. Biochem. 270:1628, 2003; Herdewijn, AntisenseNucleic Acid Develop. 10:297, 2000; Concise Encyclopedia of PolymerScience and Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990; U.S. Pat. No. 3,687,808, and similar references.

Certain nucleotide base moieties are particularly useful for increasingthe binding affinity of the dsRNA molecules of this disclosure tocomplementary targets. These include 5-substituted pyrimidines;6-azapyrimidines; and N-2, N-6, or O-6 substituted purines (including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine). Further,for example, 5-methyluridine and 5-methylcytosine substitutions areknown to increase nucleic acid duplex stability, which can be combinedwith 2′-sugar modifications (such as 2′-methoxy or 2′-methoxyethyl) orinternucleoside linkages (e.g., phosphorothioate) that provide thedesired nuclease resistance to the modified or substituted dsRNA.

In another aspect of the instant disclosure, there is provided a dsRNAthat decreases expression of one or more target gene, comprising a firststrand that is complementary to a target gene mRNA, or any combinationthereof, and a second strand that is complementary to the first strand,wherein the first and second strands form a double-stranded region ofabout 15 to about 40 base pairs; wherein at least one pyrimidine of thedsRNA is substituted with a pyrimidine nucleoside according to Formula Ior II:

wherein R¹ and R² are each independently a —H, —OH, —OCH₃, —OCH₂OCH₂CH₃,—OCH₂CH₂OCH₃, halogen, substituted or unsubstituted C₁-C₁₀ alkyl,alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino,aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl,trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted —O-allyl,—O—CH₂CH═CH₂, —O—CH═CHCH₃, substituted or unsubstituted C₂-C₁₀ alkynyl,carbamoyl, carbamyl, carboxy, carbonylamino, substituted orunsubstituted aryl, substituted or unsubstituted aralkyl, —NH₂, —NO₂, orheterocyclo group; R³ and R⁴ are each independently a hydroxyl, aprotected hydroxyl, or an internucleoside linking group; and R⁵ and R⁸are independently O or S. In certain embodiments, at least onenucleoside is according to Formula I in which R¹ is methyl and R² is —OHor R¹ is methyl, R² is —OH, and R⁸ is S. In other embodiments, theinternucleoside linking group covalently links from about 5 to about 40nucleosides.

In certain embodiments, the first and one or more second strands of adsRNA, which decreases expression of one or more target gene by RNAi andhas at least one pyrimidine substituted with a pyrimidine nucleosideaccording to Formula I or II, can anneal or hybridize together (i.e.,due to complementarity between the strands) to form at least onedouble-stranded region having a length or a combined length of about 15to about 40 base pairs. In some embodiments, the dsRNA has at least onedouble-stranded region ranging in length from about 4 base pairs toabout 10 base pairs or about 5 to about 13 base pairs or about 15 toabout 25 base pairs or about 19 to about 23 base pairs. In otherembodiments, the dsRNA has at least one double-stranded region rangingin length from about 26 to about 40 base pairs or about 27 to about 30base pairs or about 30 to about 35 base pairs. In certain embodiments,the dsRNA molecule or analog thereof has an overhang of one to fournucleotides on one or both 3′-ends, such as an overhang comprising adeoxyribonucleotide or two deoxyribonucleotides (e.g., thymidine). Insome embodiments, dsRNA molecule or analog thereof has a blunt end atone or both ends of the dsRNA. In certain embodiments, the 5′-end of thefirst or second strand is phosphorylated.

In certain embodiments, at least one R¹ is a C₁-C₅ alkyl, such as methylor ethyl. Within other exemplary embodiments of this disclosure,compounds of Formula I are a 5-alkyluridine (i.e., R¹ is alkyl, R² is—OH, and R³, R⁴, and R⁵ are as defined herein) or compounds of FormulaII are a 5-alkylcytidine (i.e., R¹ is alkyl, R² is —OH, and R³, R⁴, andR⁵ are as defined herein). In related embodiments, the 5-alkyluridine isa 5-methyluridine (also referred to as ribothymidine or ‘t’ or‘T^(r)’—i.e., R¹ is methyl and R² is —OH), and the 5-alkylcytidine is a5-methylcytidine. In other embodiments, at least one, at least three, orall uridines of the first strand of the dsRNA are replaced with5-methyluridine, or at least one, at least three, or all uridines of thesecond strand of the dsRNA are replaced with 5-methyluridine, or anycombination thereof (e.g., such changes are made on both strands). Incertain embodiments, at least one pyrimidine nucleoside of Formula I orFormula II has an R⁵ that is S or R⁸ that is S.

In further embodiments, at least one pyrimidine nucleoside of the dsRNAis a locked nucleic acid (LNA) in the form of a bicyclic sugar, whereinR² is oxygen, and the 2′O and 4′C form an oxymethylene bridge on thesame ribose ring. In a related embodiment, the LNA comprises a basesubstitution, such as a 5-methyluridine LNA or 2-thio-5-methyluridineLNA. In other embodiments, at least one, at least three, or all uridinesof the first strand of the dsRNA are replaced with 5-methyluridine or2-thioribothymidine or 5-methyluridine LNA or 2-thio-5-methyluridineLNA, or at least one, at least three, or all uridines of the secondstrand of the dsRNA are replaced with 5-methyluridine,2-thioribothymidine, 5-methyluridine LNA, 2-thio-5-methyluridine LNA, orany combination thereof (e.g., such changes are made on both strands, orsome substitutions include 5-methyluridine only, 2-thioribothymidineonly, 5-methyluridine LNA only, 2-thio-5-methyluridine LNA only, or oneor more 5-methyluridine or 2-thioribothymidine with one or more5-methyluridine LNA or 2-thio-5-methyluridine LNA).

In further embodiments, a ribose of the pyrimidine nucleoside or theinternucleoside linkage can be optionally modified. For example,compounds of Formula I or II are provided wherein R² is alkoxy, such asa 2′O-methyl substitution (e.g., which may be in addition to a5-alkyluridine or a 5-alkylcytidine, respectively). In certainembodiments, R² is selected from 2′O—(C₁-C₅) alkyl, 2′O-methyl,2′OCH₂OCH₂CH₃, 2′OCH₂CH₂OCH₃, 2′O-allyl, or 2′-fluoro. In furtherembodiments, one or more of the pyrimidine nucleosides are according toFormula I in which R¹ is methyl and R² is a 2′O—(C₁-C₅) alkyl (e.g.,2′O-methyl), or in which R¹ is methyl, R² is a 2′O—(C₁-C₅) alkyl (e.g.,2′O-methyl), and R⁵ or R⁸ is S, or any combination thereof. In otherembodiments, one or more, or at least two, pyrimidine nucleosidesaccording to Formula I or II have an R² that is not —H or —OH and isincorporated at a 3′end or 5′end and not within the gap of one or morestrands within the double-stranded region of the dsRNA molecule.

In further embodiments, a dsRNA molecule or analog thereof comprising apyrimidine nucleoside according to Formula I or Formula II in which R²is not —H or —OH and an overhang, further comprises at least two ofpyrimidine nucleosides that are incorporated either at a 3′end or a5′end or both of one strand or two strands within the double-strandedregion of the dsRNA molecule. In a related embodiment, at least one ofthe at least two pyrimidine nucleosides in which R² is not —H or —OH islocated at a 3′end or a 5′end within the double-stranded region of atleast one strand of the dsRNA molecule, and wherein at least one of theat least two pyrimidine nucleosides in which R² is not —H or —OH islocated internally within a strand of the dsRNA molecule. In stillfurther embodiments, a dsRNA molecule or analog thereof that has anoverhang has a first of the two or more pyrimidine nucleosides in whichR² is not —H or —OH that is incorporated at a 5′end within thedouble-stranded region of the sense strand of the dsRNA molecule and asecond of the two or more pyrimidine nucleosides is incorporated at a5′end within the double-stranded region of the antisense strand of thedsRNA molecule. In any of these embodiments, one or more substituted ormodified nucleotides can be a G clamp (e.g., a cytosine analog thatforms an additional hydrogen bond to guanine, such as9-(aminoethoxy)phenoxazine; see, e.g., Lin and Mateucci, 1998). In anyof these embodiments, provided the one or more pyrimidine nucleosidesare not within the gap.

In yet other embodiments, a dsRNA molecule or analog thereof of FormulaI or II according to the instant disclosure that has an overhangcomprises four or more independent pyrimidine nucleosides or four ormore independent pyrimidine nucleosides in which R² is not —H or —OH,wherein (a) a first pyrimidine nucleoside is incorporated into a 3′endwithin the double-stranded region of the sense (second) strand of thedsRNA, (b) a second pyrimidine nucleoside is incorporated into a 5′endwithin the double-stranded region of the sense (second) strand, (c) athird pyrimidine nucleoside is incorporated into a 3′end within thedouble-stranded region of the antisense (first) strand of the dsRNA, and(d) a fourth pyrimidine nucleoside is incorporated into a 5′end withinthe double-stranded region of the antisense (first) strand. In any ofthese embodiments, provided the one or more pyrimidine nucleosides arenot within the gap.

In further embodiments, a dsRNA molecule or analog thereof comprising apyrimidine nucleoside according to Formula I or Formula II in which R²is not —H or —OH and is blunt-ended, further comprises at least two ofpyrimidine nucleosides that are incorporated either at a 3′end or a5′end or both of one strand or two strands of the dsRNA molecule. In arelated embodiment, at least one of the at least two pyrimidinenucleosides in which R² is not —H or —OH is located at a 3′end or a5′end of at least one strand of the dsRNA molecule, and wherein at leastone of the at least two pyrimidine nucleosides in which R² is not —H or—OH is located internally within a strand of the dsRNA molecule. Instill further embodiments, a dsRNA molecule or analog thereof that isblunt-ended has a first of the two or more pyrimidine nucleosides inwhich R² is not —H or —OH that is incorporated at a 5′end of the sensestrand of the dsRNA molecule and a second of the two or more pyrimidinenucleosides is incorporated at a 5′end of the antisense strand of thedsRNA molecule. In any of these embodiments, provided the one or morepyrimidine nucleosides are not within the gap.

In yet other embodiments, a dsRNA molecule comprising a pyrimidinenucleoside according to Formula I or II and that is blunt-endedcomprises four or more independent pyrimidine nucleosides or four ormore independent pyrimidine nucleosides in which R² is not —H or —OH,wherein (a) a first pyrimidine nucleoside is incorporated into a 3′endwithin the double-stranded region of the sense (second) strand of thedsRNA, (b) a second pyrimidine nucleoside is incorporated into a 5′endwithin the double-stranded region of the sense (second) strand, (c) athird pyrimidine nucleoside is incorporated into a 3′end within thedouble-stranded region of the antisense (first) strand of the dsRNA, and(d) a fourth pyrimidine nucleoside is incorporated into a 5′end withinthe double-stranded region of the antisense (first) strand. In any ofthese embodiments, provided the one or more pyrimidine nucleosides arenot within the gap.

In still further embodiments, a dsRNA molecule or analog thereof ofFormula I or II according to the instant disclosure further comprises aterminal cap substituent on one or both ends of the first strand orsecond strand, such as an alkyl, abasic, deoxy abasic, glyceryl,dinucleotide, acyclic nucleotide, inverted deoxynucleotide moiety, orany combination thereof. In further embodiments, one or moreinternucleoside linkage can be optionally modified. For example, a dsRNAmolecule or analog thereof of Formula I or II according to the instantdisclosure wherein at least one internucleoside linkage is modified to aphosphorothioate, chiral phosphorothioate, phosphorodithioate,phosphotriester, aminoalkylphosphotriester, methyl phosphonate, alkylphosphonate, 3′-alkylene phosphonate, 5′-alkylene phosphonate, chiralphosphonate, phosphonoacetate, thiophosphonoacetate, phosphinate,phosphoramidate, 3′-amino phosphoramidate, aminoalkylphosphoramidate,thionophosphoramidate, thionoalkylphosphonate,thionoalkylphosphotriester, selenophosphate, boranophosphate linkage, orany combination thereof.

In still another embodiment, provided is a nicked or gapped dsRNAmolecule (ndsRNA or gdsRNA, respectively) that decreases expression ofone or more target gene by RNAi, which comprises a first strand that iscomplementary to a target gene mRNA, or any combination thereof, and twoor more second strands that are complementary to the first strand,wherein the first and at least one of the second strands form anon-overlapping double-stranded region of about 5 to about 13 basepairs. Any of the aforementioned substitutions or modifications iscontemplated within this embodiment as well.

In another exemplary of this disclosure, the dsRNAs comprise at leasttwo or more substituted pyrimidine nucleosides can each be independentlyselected wherein R¹ comprises any chemical modification or substitutionas contemplated herein, for example, an alkyl (e.g., methyl), halogen,hydroxy, alkoxy, nitro, amino, trifluoromethyl, cycloalkyl,(cycloalkyl)alkyl, alkanoyl, alkanoyloxy, aryl, aroyl, aralkyl, nitrile,dialkylamino, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl,alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, carboxyalkyl,alkoxyalkyl, carboxy, carbonyl, alkanoylamino, carbamoyl, carbonylamino,alkylsulfonylamino, or heterocyclo group. When two or more modifiedribonucleotides are present, each modified ribonucleotide can beindependently modified to have the same, or different, modification orsubstitution at R¹ or R².

In other detailed embodiments, one or more substituted pyrimidinenucleosides according to Formula I or II can be located at anyribonucleotide position, or any combination of ribonucleotide positions,on either or both of the sense and antisense strands of a dsRNA moleculeof this disclosure, including at one or more multiple terminal positionsas noted above, or at any one or combination of multiple non-terminal(“internal”) positions. In this regard, each of the sense and antisensestrands can incorporate about 1 to about 6 or more of the substitutedpyrimidine nucleosides.

In certain embodiments, when two or more substituted pyrimidinenucleosides are incorporated within a dsRNA of this disclosure, at leastone of the substituted pyrimidine nucleosides will be at a 3′ or 5′endof one or both strands, and in certain embodiments at least one of thesubstituted pyrimidine nucleosides will be at a 5′end of one or bothstrands. In other embodiments, the substituted pyrimidine nucleosidesare located at a position corresponding to a position of a pyrimidine inan unmodified dsRNA that is constructed as a homologous sequence fortargeting a cognate mRNA, as described herein.

In addition, the terminal structure of the dsRNAs of this disclosure mayhave a stem-loop structure in which ends of one side of the dsRNAmolecule are connected by a linker nucleic acid, e.g., a linker RNA. Thelength of the double-stranded region (stem-loop portion) can be, forexample, about 15 to about 49 basepairs (bp), about 15 to about 35 bp,or about 21 to about 30 bp long. Alternatively, the length of thedouble-stranded region that is a final transcription product of dsRNAsto be expressed in a target cell may be, for example, approximatelyabout 15 to about 49 bp, about 15 to about 35 bp, or about 21 to about30 bp long. When linker segments are employed, there is no particularlimitation in the length of the linker as long as it does not hinderpairing of the stem portion. For example, for stable pairing of the stemportion and suppression of recombination between DNAs coding for thisportion, the linker portion may have a clover-leaf tRNA structure. Evenif the linker has a length that would hinder pairing of the stemportion, it is possible, for example, to construct the linker portion toinclude introns so that the introns are excised during processing of aprecursor RNA into mature RNA, thereby allowing pairing of the stemportion. In the case of a stem-loop dsRNA, either end (head or tail) ofRNA with no loop structure may have a low molecular weight RNA. Asdescribed above, these low molecular weight RNAs may include a naturalRNA molecule, such as tRNA, rRNA or viral RNA, or an artificial RNAmolecule.

A dsRNA molecule may be comprised of a circular nucleic acid molecule,wherein the dsRNA is about 38 to about 70 nucleotides in length havingfrom about 18 to about 23 base pairs (e.g., about 19 to about 21)wherein the circular oligonucleotide forms a dumbbell shaped structurehaving about 19 base pairs and two loops. In certain embodiments, acircular dsRNA molecule contains two loop motifs, wherein one or bothloop portions of the dsRNA molecule is biodegradable. For example, acircular dsRNA molecule of this disclosure is designed such thatdegradation of the loop portions of the dsRNA molecule in vivo cangenerate a double-stranded dsRNA molecule with 3′terminal overhangs,such as 3′terminal nucleotide overhangs comprising from about 1 to about4 (unpaired) nucleotides.

Substituting pyrimidine nucleosides into a dsRNA according to thisdisclosure can be chosen to increase resistance to enzymaticdegradation, such as exonucleolytic degradation, including5′exonucleolytic or 3′exonucleolytic degradation. As such, the dsRNAsdescribed herein will exhibit significant resistance to enzymaticdegradation compared to a corresponding dsRNA having standardnucleotides, and will thereby possess greater stability, increasedhalf-life, and greater bioavailability in physiological environments(e.g., when introduced into a eukaryotic target cell). In addition toincreasing resistance of the substituted or modified dsRNAs toexonucleolytic degradation, the incorporation of one or more pyrimidinenucleosides according to Formula I or II will render dsRNAs can makethese molecules more stable and bioavailable than otherwise identicaldsRNAs that do not include the substitutions or modifications. Inrelated aspects of this disclosure, dsRNA substitutions or modificationsdescribed herein are chosen to improve stability of a modified dsRNA foruse within research, diagnostic and treatment methods wherein themodified dsRNA is contacted with a biological sample, for example, amammalian cell, intracellular compartment, serum or other extracellularfluid, tissue, or other in vitro or in vivo physiological compartment orenvironment. In one embodiment, diagnosis is performed on an isolatedbiological sample. In another embodiment, the diagnostic method isperformed in vitro. In a further embodiment, the diagnostic method isnot performed (directly) on a human or animal body.

In addition to increasing stability of substituted or modified dsRNAs,incorporation of one or more pyrimidine nucleosides according to FormulaI or II in a dsRNA designed for gene silencing can be used to yieldadditional desired functional results, including increasing a meltingpoint of a substituted or modified dsRNA compared to a corresponding,unmodified dsRNA. By thus increasing a dsRNA melting point, the subjectsubstitutions or modifications will often block or reduce the occurrenceor extent of partial dehybridization of the substituted or modifieddsRNA (that would ordinarily occur and render the unmodified dsRNA morevulnerable to degradation by certain exonucleases), thereby increasingthe stability of the substituted or modified dsRNA.

In another aspect of this disclosure, the mdRNA structure can be used toreduce off-target effects, which can be improved with substitutions ormodifications described herein, when the mdRNA are contacted with abiological sample (e.g., when introduced into a target eukaryotic cellhaving specific, and non-specific mRNA species present as potentialspecific and non-specific targets). Similarly, the mdRNA structure canbe used to reduce interferon activation, which can be improved withsubstitutions or modifications described herein, when the mdRNA iscontacted with a biological sample, for example, when introduced into aeukaryotic cell. Hence, substituted or modified dsRNAs (mdRNAs)according to this disclosure are employed in methods of gene silencing,wherein the substituted or modified dsRNAs exhibit reduced orundetectable off-target effects or reduced interferon response comparedto a corresponding dsRNA lack a nick or gap or modification.

In further embodiments, dsRNAs of this disclosure can comprise one ormore sense (second) strand that is homologous or corresponds to asequence of a target gene and an antisense (first) strand that iscomplementary to the sense strand and a sequence of the target gene. Inexemplary embodiments, at least one strand of the dsRNA incorporates oneor more pyrimidines substituted according to Formula I or II (e.g.,wherein the pyrimidine is replaced by one ore more 5-methyluridines or2-thioribothymidines, the ribose is modified to incorporate one or more2′-O-methyl substitutions, or any combination thereof). These and othermultiple substitutions or modifications according to Formula I or II canbe introduced into one or more pyrimidines, or into any combination andup to all pyrimidines present in one or both strands of a dsRNA, so longas the dsRNA retains RNAi activity.

In any of the embodiments described herein, the dsRNA may includemultiple modifications. For example, a dsRNA having at least oneribothymidine or 2-thioribothymidine may further comprise at least oneLNA, 2′-methoxy, 2′-fluoro, 2′-deoxy, phosphorothioate linkage, aninverted base terminal cap, or any combination thereof. In certainembodiments, a dsRNA will have from one to all uridines substituted withribothymidine and have up to about 75% LNA substitutions. In otherembodiments, a dsRNA will have from one to all uridines substituted withribothymidine and have up to about 75% 2′-methoxy substitutions (and notat the Argonaute cleavage site). In still other embodiments, a dsRNAwill have from one to all uridines substituted with ribothymidine andhave up to about 100% 2′-fluoro substitutions. In further embodiments, adsRNA will have from one to all uridines substituted with ribothymidineand have up to about 75% 2′-deoxy substitutions. In further embodiments,a dsRNA will have up to about 75% LNA substitutions and have up to about75% 2′-methoxy substitutions. In still other embodiments, a dsRNA willhave up to about 75% LNA substitutions and have up to about 100%2′-fluoro substitutions. In further embodiments, a dsRNA will have up toabout 75% LNA substitutions and have up to about 75% 2′-deoxysubstitutions. In further embodiments, a dsRNA will have up to about 75%2′-methoxy substitutions and have up to about 100% 2′-fluorosubstitutions. In further embodiments, a dsRNA will have up to about 75%2′-methoxy substitutions and have up to about 75% 2′-deoxysubstitutions. In further embodiments, a dsRNA will have up to about100% 2′-fluoro substitutions and have up to about 75% 2′-deoxysubstitutions.

In further multiple modification embodiments, a dsRNA will have from oneto all uridines substituted with ribothymidine, up to about 75% LNAsubstitutions, and up to about 75% 2′-methoxy substitutions. In stillfurther embodiments, a dsRNA will have from one to all uridinessubstituted with ribothymidine, up to about 75% LNA substitutions, andup to about 100% 2′-fluoro substitutions. In further embodiments, adsRNA will have from one to all uridines substituted with ribothymidine,up to about 75% LNA substitutions, and up to about 75% 2′-deoxysubstitutions. In further embodiments, a dsRNA will have from one to alluridines substituted with ribothymidine, up to about 75% 2′-methoxysubstitutions, and up to about 75% 2′-fluoro substitutions. In furtherembodiments, a dsRNA will have from one to all uridines substituted withribothymidine, up to about 75% 2′-methoxy substitutions, and up to about75% 2′-deoxy substitutions. In further embodiments, a dsRNA will havefrom one to all uridines substituted with ribothymidine, up to about100% 2′-fluoro substitutions, and up to about 75% 2′-deoxysubstitutions. In yet further embodiments, a dsRNA will have from one toall uridines substituted with ribothymidine, up to about 75% LNAsubstitutions, up to about 75% 2′-methoxy, up to about 100% 2′-fluoro,and up to about 75% 2′-deoxy substitutions. In other embodiments, adsRNA will have up to about 75% LNA substitutions, up to about 75%2′-methoxy substitutions, and up to about 100% 2′-fluoro substitutions.In further embodiments, a dsRNA will have up to about 75% LNAsubstitutions, up to about 75% 2′-methoxy substitutions, and up to about75% 2′-deoxy substitutions. In further embodiments, a dsRNA will have upto about 75% LNA substitutions, up to about 100% 2′-fluorosubstitutions, and up to about 75% 2′-deoxy substitutions. In stillfurther embodiments, a dsRNA will have up to about 75% 2′-methoxy, up toabout 100% 2′-fluoro, and up to about 75% 2′-deoxy substitutions.

In any of these multiple modification embodiments, the dsRNA may furthercomprise up to 100% phosphorothioate internucleoside linkages, from oneto ten or more inverted base terminal caps, or any combination thereof.Additionally, any of these multiple modification embodiments may havethese multiple modifications on one strand, two strands, three strands,a plurality of strand, or all strands. Finally, in any of these multiplemodification dsRNA, the dsRNA must retain gene silencing activity.

Within certain aspects, the present disclosure provides dsRNA thatdecreases expression of one or more target gene by RNAi, andcompositions comprising one or more dsRNA, wherein at least one dsRNAcomprises one or more universal-binding nucleotide(s) in the first,second or third position in the anti-codon of the antisense strand ofthe dsRNA duplex and wherein the dsRNA is capable of specificallybinding to one or more target sequence, such as an RNA expressed by atarget cell. In cases wherein the sequence of a target RNA includes oneor more single nucleotide substitutions, dsRNA comprising auniversal-binding nucleotide retains its capacity to specifically bind atarget RNA, thereby mediating gene silencing and, as a consequence,overcoming escape of the target from dsRNA-mediated gene silencing.Non-limiting examples of universal-binding nucleotides that may besuitably employed in the compositions and methods disclosed hereininclude inosine, 1-β-D-ribofuranosyl-5-nitroindole, and1-β-D-ribofuranosyl-3-nitropyrrole. For the purpose of the presentdisclosure, a universal-binding nucleotide is a nucleotide that can forma hydrogen bonded nucleotide pair with more than one nucleotide type.

Non-limiting examples for the above compositions includes modifying theanti-codons for tyrosine (AUA) or phenylalanine (AAA or GAA), cysteine(ACA or GCA), histidine (AUG or GUG), asparagine (AUU or GUU),isoleucine (UAU) and aspartate (AUC or GUC) within the anti-codon of theantisense strand of the dsRNA molecule.

For example, within certain embodiments, the isoleucine anti-codon UAU,for which AUA is the cognate codon, may be modified such that thethird-position uridine (U) nucleotide is substituted with theuniversal-binding nucleotide inosine I to create the anti-codon UAI.Inosine is an exemplary universal-binding nucleotide that can pair withan adenosine (A), uridine (U), and cytidine (C) nucleotide, but notguanosine (G). This modified anti-codon UAI increases thespecific-binding capacity of the dsRNA molecule and thus permits thedsRNA to pair with mRNAs having any one of AUA, UUA, and CUA in thecorresponding position of the coding strand thereby expanding the numberof available RNA degradation targets to which the dsRNA may specificallybind.

Alternatively, the anti-codon AUA may also or alternatively be modifiedby substituting a universal-binding nucleotide in the third or secondposition of the anti-codon such that the anti-codon(s) represented byUAI (third position substitution) or UIU (second position substitution)to generate dsRNA that are capable of specifically binding to AUA, CUAand UUA and AAA, ACA and AUA.

In certain aspects, dsRNA disclosed herein can include between about 1universal-binding nucleotide and about 10 universal-binding nucleotides.Within certain aspects, the presently disclosed dsRNA may comprise asense strand that is homologous to a sequence of one or more target geneand an antisense strand that is complementary to the sense strand, withthe proviso that at least one nucleotide of the antisense strand of theotherwise complementary dsRNA duplex is replaced by one or moreuniversal-binding nucleotide.

By way of background, within the silencing complex, the dsRNA moleculeis positioned so that it can interact or bind to a target RNA. The RISCwill encounter thousands of different RNAs that are in a typical cell atany given moment. But, the dsRNA loaded in RISC will specifically annealwith a target RNA that has close complementarity with the antisense ofthe dsRNA molecule. So, unlike an interferon response to a viralinfection, the silencing complex is highly selective in identifying atarget RNA. RISC cleaves the captured target RNA strand and releases thetwo pieces of the RNA (now rendered incapable of directing proteinsynthesis) and moves on. RISC itself stays intact and is capable offinding and cleaving additional target RNA molecules.

It will be understood that, regardless of the position at which the oneor more universal-binding nucleotide is substituted, the dsRNA moleculeis capable of binding to a target gene and one or more variant(s)thereof thereby facilitating the degradation of the target gene orvariant thereof via Dicer or RISC. Thus, the dsRNA of the presentdisclosure are suitable for introduction into cells to mediate targetedpost-transcriptional gene silencing of one or more target gene orvariants thereof. When a dsRNA is inserted into a cell, the dsRNA duplexis then unwound, and the antisense strand anneals with mRNA to form aDicer substrate or the antisense strand is loaded into an assembly ofproteins to form the RNA-induced silencing complex (RISC).

Synthesis of Gapped or Nicked dsRNA Molecules

Exemplary molecules of the instant disclosure are recombinantlyproduced, chemically synthesized, or a combination thereof.Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example, as described in Caruthers etal., Methods in Enzymol. 211:3, 1992; Thompson et al., PCT PublicationNo. WO 99/54459, Wincott et al., Nucleic Acids Res. 23:2677, 1995;Wincott et al., Methods Mol. Bio. 74:59, 1997; Brennan et al.,Biotechnot Bioeng. 61:33-45, 1998; and Brennan, U.S. Pat. No. 6,001,311.Synthesis of RNA, including certain dsRNA molecules and analogs thereofof this disclosure, can be made using the procedure as described inUsman et al., J. Am. Chem. Soc. 109:7845, 1987; Scaringe et al., NucleicAcids Res. 18:5433, 1990; and Wincott et al., Nucleic Acids Res.23:2677-2684, 1995; Wincott et al., Methods Mol. Bio. 74:59, 1997.

In certain embodiments, the nucleic acid molecules of the presentdisclosure can be synthesized separately and joined togetherpost-synthetically, for example, by ligation (Moore et al., Science256:9923, 1992; Draper et al., PCT Publication No. WO 93/23569;Shabarova et al., Nucleic Acids Res. 19:4247, 1991; Bellon et al.,Nucleosides & Nucleotides 16:951, 1997; Bellon et al., BioconjugateChem. 8:204, 1997), or by hybridization following synthesis ordeprotection.

In further embodiments, dsRNAs of this disclosure that decreaseexpression of one or more target family gene by RNAi can be made assingle or multiple transcription products expressed by a polynucleotidevector encoding one or more dsRNAs and directing their expression withinhost cells. In these embodiments the double-stranded portion of a finaltranscription product of the dsRNAs to be expressed within the targetcell can be, for example, about 5 to about 40 bp, about 15 to about 24bp, or about 25 to about 40 bp long. Within exemplary embodiments,double-stranded portions of dsRNAs, in which two or more strands pairup, are not limited to completely paired nucleotide segments, and maycontain non-pairing portions due to a mismatch (the correspondingnucleotides are not complementary), bulge (lacking in the correspondingcomplementary nucleotide on one strand), overhang, or the like.Non-pairing portions can be contained to the extent that they do notinterfere with dsRNA formation and function. In certain embodiments, a“bulge” may comprise 1 to 2 non-pairing nucleotides, and thedouble-stranded region of dsRNAs in which two strands pair up maycontain from about 1 to 7, or about 1 to 5 bulges. In addition,“mismatch” portions contained in the double-stranded region of dsRNAsmay include from about 1 to 7, or about 1 to 5 mismatches. In otherembodiments, the double-stranded region of dsRNAs of this disclosure maycontain both bulge and mismatched portions in the approximate numericalranges specified herein.

A dsRNA or analog thereof of this disclosure may be further comprised ofa nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linkerthat joins the sense region of the dsRNA to the antisense region of thedsRNA. In one embodiment, a nucleotide linker can be a linker of morethan about 2 nucleotides length up to about 10 nucleotides in length. Inanother embodiment, the nucleotide linker can be a nucleic acid aptamer.By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleicacid molecule that binds specifically to a target molecule wherein thenucleic acid molecule has sequence that comprises a sequence recognizedby the target molecule in its natural setting. Alternately, an aptamercan be a nucleic acid molecule that binds to a target molecule whereinthe target molecule does not naturally bind to a nucleic acid. Thetarget molecule can be any molecule of interest. For example, theaptamer can be used to bind to a ligand-binding domain of a protein,thereby preventing interaction of the naturally occurring ligand withthe protein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art (see, e.g., Gold et al., Annu.Rev. Biochem. 64:763, 1995; Brody and Gold, J. Biotechnol. 74:5, 2000;Sun, Curr. Opin. Mol. Ther. 2:100, 2000; Kusser, J. Biotechnol. 74:27,2000; Hermann and Patel, Science 287:820, 2000; and Jayasena, ClinicalChem. 45:1628, 1999).

A non-nucleotide linker may be comprised of an abasic nucleotide,polyether, polyamine, polyamide, peptide, carbohydrate, lipid,polyhydrocarbon, or other polymeric compounds (e.g., polyethyleneglycols such as those having between 2 and 100 ethylene glycol units).Specific examples include those described by Seela and Kaiser, NucleicAcids Res. 18:6353, 1990; Seela and Kaiser, Nucleic Acids Res. 15:3113,1987; Cload and Schepartz, J. Am. Chem. Soc. 113:6324, 1991; Richardsonand Schepartz, J. Am. Chem. Soc. 113:5109, 1991; Ma et al., NucleicAcids Res. 21:2585, 1993; Ma et al., Biochemistry 32:1751, 1993; Durandet al., Nucleic Acids Res. 18:6353, 1990; McCurdy et al., Nucleosides &Nucleotides 10:287, 1991; Jaschke et al., Tetrahedron Lett. 34:301,1993; Ono et al., Biochemistry 30:9914, 1991; Arnold et al.,International Publication No. WO 89/02439; Usman et al., PCT PublicationNo. WO 95/06731; Dudycz et al., PCT Publication No. WO 95/11910; andFerentz and Verdine, J. Am. Chem. Soc. 113:4000, 1991. The synthesis ofa dsRNA molecule of this disclosure, which can be further modified,comprises: (a) synthesis of two complementary strands of the dsRNAmolecule; and (b) annealing the two complementary strands together underconditions suitable to obtain a dsRNA molecule. In another embodiment,synthesis of the two complementary strands of a dsRNA molecule is bysolid phase oligonucleotide synthesis. In yet another embodiment,synthesis of the two complementary strands of a dsRNA molecule is bysolid phase tandem oligonucleotide synthesis.

Chemically synthesizing nucleic acid molecules with substitutions ormodifications (base, sugar, phosphate, or any combination thereof) canprevent their degradation by serum ribonucleases, which may lead toincreased potency. See, for example, Eckstein et al., PCT PublicationNo. WO 92/07065; Perrault et al., Nature 344:565, 1990; Pieken et al.,Science 253:314, 1991; Usman and Cedergren, Trends in Biochem. Sci.17:334, 1992; Usman et al., Nucleic Acids Symp. Ser. 31:163, 1994;Beigelman et al., J. Biol. Chem. 270:25702, 1995; Burgin et al.,Biochemistry 35:14090, 1996; Burlina et al., Bioorg. Med. Chem.5:1999-2010, 1997; Thompson et al., Karpeisky et al., Tetrahedron Lett.39:1131, 1998; Earnshaw and Gait, Biopolymers (Nucleic Acid Sciences)48:39-55, 1998; Verma and Eckstein, Annu. Rev. Biochem. 67:99-134, 1998;Herdewijn, Antisense Nucleic Acid Drug Dev. 10:297, 2000; Kurreck, Eur.J. Biochem. 270:1628, 2003; Dorsett and Tuschl, Nature Rev. Drug Discov.3:318, 2004; Rossi et al., PCT Publication No. WO 91/03162; Usman etal., PCT Publication No. WO 93/15187; Beigelman et al., PCT PublicationNo. WO 97/26270; Woolf et al., PCT Publication No. WO 98/13526; Sproat,U.S. Pat. No. 5,334,711; Usman et al., U.S. Pat. No. 5,627,053;Beigelman et al., U.S. Pat. No. 5,716,824; Otvos et al., U.S. Pat. No.5,767,264; Gold et al., U.S. Pat. No. 6,300,074. Each of the abovereferences discloses various substitutions and chemical modifications tothe base, phosphate, or sugar moieties of nucleic acid molecules, whichcan be used in the dsRNAs described herein. For example,oligonucleotides can be modified at the sugar moiety to enhancestability or prolong biological activity by increasing nucleaseresistance. Representatives of such sugar modifications include 2′amino,2′C-allyl, 2′-fluoro, 2′-O-methyl, 2′-O-allyl, or 2′-deoxy. Hence, dsRNAmolecules of the instant disclosure can be modified to increase nucleaseresistance or duplex stability while substantially retaining or havingenhanced RNAi activity as compared to unmodified dsRNA.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability or enhance biological activity by modification with nucleaseresistant groups, for example, 2′amino, 2′C-allyl, 2′fluoro, 2′O-methyl,2′O-allyl, 2′-H, nucleotide base modifications. For a review, see Usmanand Cedergren, TIBS 17:34, 1992; Usman et al., Nucleic Acids Symp. Ser.31:163, 1994; Burgin et al., Biochemistry 35:14090, 1996. Sugarmodification of nucleic acid molecules have been extensively describedin the art (see Eckstein et al., PCT Publication No. WO 92/07065;Perrault et al., Nature 344:565-568, 1990; Pieken et al., Science253:314-317, 1991; Usman and Cedergren, Trends in Biochem. Sci.17:334-339, 1992; Usman et al., PCT Publication No. WO 93/15187; Sproat,U.S. Pat. No. 5,334,711 and Beigelman et cd., J. Biol. Chem. 270:25702,1995; Beigelman et al., PCT Publication No. WO 97/26270; Beigelman etal., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053;Woolf et al., PCT Publication No. WO 98/13526; Thompson et al.,Karpeisky et al., Tetrahedron Lett. 39:1131, 1998; Earnshaw and Gait,Biopolymers (Nucleic Acid Sciences) 48:39-55, 1998; Verma and Eckstein,Annu. Rev. Biochem. 67:99-134, 1998; and Burlina et al., Bioorg. Med.Chem. 5:1999-2010, 1997. Such publications describe general methods andstrategies to determine the location of incorporation of sugar, base orphosphate modifications and the like into nucleic acid molecules withoutmodulating catalysis. In view of such teachings, similar modificationscan be used as described herein to modify the dsRNA molecules of theinstant disclosure so long as the ability of dsRNA molecules to promoteRNAi in cells is not significantly inhibited.

In one embodiment, this disclosure features substituted or modifieddsRNA molecules, such as phosphate backbone modifications comprising oneor more phosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417, 1995; and Mesmaeker et al., ACS, 24-39, 1994.

In another embodiment, a conjugate molecule can be optionally attachedto a dsRNA or analog thereof that decreases expression of one or moretarget gene by RNAi. For example, such conjugate molecules may bepolyethylene glycol, human serum albumin, or a ligand for a cellularreceptor that can, for example, mediate cellular uptake. Examples ofspecific conjugate molecules contemplated by the instant disclosure thatcan be attached to a dsRNA or analog thereof of this disclosure aredescribed in Vargeese et al., U.S. Patent Application Publication No.2003/0130186, published Jul. 10, 2003, and U.S. Patent ApplicationPublication No. 2004/0110296, published Jun. 10, 2004. In anotherembodiment, a conjugate molecule is covalently attached to a dsRNA oranalog thereof that decreases expression of one or more target gene byRNAi via a biodegradable linker. In certain embodiments, a conjugatemolecule can be attached at the 3′end of either the sense strand, theantisense strand, or both strands of a dsRNA molecule provided herein.In another embodiment, a conjugate molecule can be attached at the 5′endof either the sense strand, the antisense strand, or both strands of thedsRNA or analog thereof. In yet another embodiment, a conjugate moleculeis attached both the 3′end and 5′end of either the sense strand, theantisense strand, or both strands of a dsRNA molecule, or anycombination thereof. In further embodiments, a conjugate molecule ofthis disclosure comprises a molecule that facilitates delivery of adsRNA or analog thereof into a biological system, such as a cell. Aperson of skill in the art can screen dsRNA of this disclosure havingvarious conjugates to determine whether the dsRNA-conjugate complexpossesses improved properties (e.g., pharmacokinetic profile,bioavailability, stability) while maintaining the ability to mediateRNAi in, for example, an animal model as described herein or generallyknown in the art.

Methods for Selecting dsRNA Molecules Specific for a Target Sequence

As indicated above, the present disclosure also provides methods forselecting dsRNA and analogs thereof capable of specifically binding toone or more target gene while being not specifically binding orminimally binding to non-target genes. The selection process disclosedherein is useful, for example, in eliminating dsRNA analogs that arecytotoxic due to non-specific binding to, and subsequent degradation of,one or more non-target genes.

Methods of the present disclosure do not require a priori knowledge ofthe nucleotide sequence of every possible gene variant targeted by thedsRNA or analog thereof. In one embodiment, the nucleotide sequence ofthe dsRNA is selected from a conserved region or consensus sequence ofone or more target gene. In another embodiment, the nucleotide sequenceof the dsRNA may be selectively or preferentially targeted to a certainsequence contained within an mRNA splice variant of a target gene.

In certain embodiments, methods are provided for selecting one or moredsRNA molecule that decreases expression of one or more target gene byRNAi, comprising a first strand that is complementary to a target genemRNA, or any combination thereof, and a second strand that iscomplementary to the first strand, wherein the first and second strandsform a double-stranded region of about 15 to about 40 base pairs, andwherein at least one uridine of the dsRNA molecule is replaced with a5-methyluridine or 2-thioribothymidine, which methods employ“off-target” profiling whereby one or more dsRNA provided herein iscontacted with a cell, either in vivo or in vitro, and total target mRNAis collected for use in probing a microarray comprising oligonucleotideshaving one or more nucleotide sequence from a panel of known genes,including non-target genes (e.g., interferon). The “off-target” profileof the dsRNA provided herein is quantified by determining the number ofnon-target genes having reduced expression levels in the presence of thecandidate dsRNAs. The existence of “off target” binding indicates adsRNA provided herein that is capable of specifically binding to one ormore non-target gene messages. In certain embodiments, a dsRNA asprovided herein applicable to therapeutic use will exhibit a greaterstability, minimal interferon response, and little or no “off-target”binding.

Still further embodiments provide methods for selecting more efficaciousdsRNA by using one or more reporter gene constructs comprising aconstitutive promoter, such as a cytomegalovirus (CMV) orphosphoglycerate kinase (PGK) promoter, operably fused to, and capableof altering the expression of one or more reporter genes, such as aluciferase, chloramphenicol (CAT), or β-galactosidase, which, in turn,is operably fused in-frame with a dsRNA (such as one having a lengthbetween about 15 base-pairs and about 40 base-pairs or from about 5nucleotides to about 24 nucleotides, or about 25 nucleotides to about 40nucleotides) that contains one or more target sequence, as providedherein.

Individual reporter gene expression constructs may be co-transfectedwith one or more dsRNA or analog thereof. The capacity of a given dsRNAto reduce the expression level of a target gene may be determined bycomparing the measured reporter gene activity in cells transfected withor without a dsRNA molecule of interest.

Certain embodiments disclosed herein provide methods for selecting oneor more modified dsRNA molecule(s) that employ the step of predictingthe stability of a dsRNA duplex. In some embodiments, such a predictionis achieved by employing a theoretical melting curve wherein a highertheoretical melting curve indicates an increase in dsRNA duplexstability and a concomitant decrease in cytotoxic effects.Alternatively, stability of a dsRNA duplex may be determined empiricallyby measuring the hybridization of a single RNA analog strand asdescribed herein to a complementary target gene within, for example, apolynucleotide array. The melting temperature (i.e., the T_(m) value)for each modified RNA and complementary RNA immobilized on the array canbe determined, and from this T_(m) value, the relative stability of asubstituted or modified RNA pairing with a complementary RNA moleculecan be determined.

For example, for universal-binding nucleotide, Kawase et al. (NucleicAcids Res. 14:7727, 1986) have described an analysis of thenucleotide-pairing properties of Di (inosine) to A, C, G, and T, whichwas achieved by measuring the hybridization of oligonucleotides (ODNs)with Di in various positions to complementary sets of ODNs made as anarray. The relative strength of nucleotide-pairing is I-C>I-A>I-G≈I-T.Generally, Di containing duplexes showed lower T_(m) values whencompared to the corresponding WC nucleotide pair. The stabilization ofDi by pairing was in order of Dc>Da>Dg>Dt>Du. From the thermodynamicvalues calculated using Van't Hoff plots according to a two state model,Kawase et al. conclude that the sequence of purine-pyrimidine is favoredin double strand formation due to nucleotide stacking. For instance theduplex formation of XY=AT is a more favored formation than an XY=CG andTA. As a person of skill in the art would understand, althoughuniversal-binding nucleotides are used herein as an example ofdetermining stability (i.e., the T_(m) value), other nucleotidesubstitutions (e.g., 5-methyluridine for uridine) or furthermodifications (e.g., a ribose modification at the 2′-position) can alsobe evaluated by these or similar methods.

Within certain embodiments, methods disclosed herein comprise the stepsof (a) designing or synthesizing a suitable dsRNA for RNAi genesilencing of one or more target gene, wherein the dsRNA comprises atleast three strands and optionally at least one modification orsubstitution (such as a 5-methyluridine, LNA, 2′-methoxy, 2′-fluoro,phosphorothioate, or any combination thereof); and (b) contacting a cellexpressing one or more target protein with the dsRNA, wherein the dsRNAis capable of specifically binding to one or more target mRNA or gene,thereby reducing expression of one or more target members.

Any of these methods of identifying dsRNA of interest can also be usedto examine a dsRNA that decreases expression of one or more target geneby RNA interference, comprising a first strand that is complementary toa target mRNA, or any combination thereof, and a second and third strandthat have non-overlapping complementarity to the first strand, whereinthe first and at least one of the second or third strand form adouble-stranded region of about 5 to about 13 base pairs; wherein atleast one pyrimidine of the dsRNA comprises a pyrimidine nucleosideaccording to Formula I or II:

wherein R¹ and R² are each independently a —OH, —OCH₃, —OCH₂OCH₂CH₃,—OCH₂CH₂OCH₃, halogen, substituted or unsubstituted C₁-C₁₀ alkyl,alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino,aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl,trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted —O-allyl,—O—CH₂CH═CH₂, —O—CH═CHCH₃, substituted or unsubstituted C₂-C₁₀ alkynyl,carbamoyl, carbamyl, carboxy, carbonylamino, substituted orunsubstituted aryl, substituted or unsubstituted aralkyl, —NH₂, —NO₂,—C≡N, or heterocyclo group; R³ and R⁴ are each independently a hydroxyl,a protected hydroxyl, or an internucleoside linking group; and R⁵ and R⁸are independently O or S. In certain embodiments, at least onenucleoside is according to Formula I in which R¹ is methyl and R² is—OH, or R¹ is methyl, R² is —OH, and R⁸ is S. In other embodiments, theinternucleoside linking group covalently links from about 5 to about 40nucleosides.

Compositions and Methods of Use

As set forth herein, dsRNA of the instant disclosure are designed totarget one or more target gene (including one or more mRNA splicevariants thereof) that is expressed at an elevated level or continues tobe expressed when it should not, and is a causal or contributing factorassociated with, for example, a hyperproliferative, angiogenic, orinflammatory disease, state, or adverse condition. In this context, adsRNA or analog thereof of this disclosure will effectively downregulate expression of one or more target gene to levels that prevent,alleviate, or reduce the severity or recurrence of one or moreassociated disease symptoms. Alternatively, for various distinct diseasemodels in which expression of one or more target gene is not necessarilyelevated as a consequence or sequel of disease or other adversecondition, down regulation of one or more target gene will nonethelessresult in a therapeutic result by lowering gene expression. Furthermore,dsRNAs of this disclosure may be targeted to reduce expression of one ormore target gene, which can result in upregulation of a “downstream”gene whose expression is negatively regulated, directly or indirectly,by one or more target protein. The dsRNA molecules of the instantdisclosure comprise useful reagents and can be used in methods for avariety of therapeutic, diagnostic, target validation, genomicdiscovery, genetic engineering, and pharmacogenomic applications.

In certain embodiments, aqueous suspensions contain dsRNA of thisdisclosure in admixture with suitable excipients, such as suspendingagents or dispersing or wetting agents. Exemplary suspending agentsinclude sodium carboxymethylcellulose, methylcellulose,hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia. Representative dispersing or wetting agentsinclude naturally-occurring phosphatides (e.g., lecithin), condensationproducts of an alkylene oxide with fatty acids (e.g., polyoxyethylenestearate), condensation products of ethylene oxide with long chainaliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides (e.g., polyethylene sorbitan monooleate). Incertain embodiments, the aqueous suspensions can optionally contain oneor more preservatives (e.g., ethyl or n-propyl-p-hydroxybenzoate), oneor more coloring agents, one or more flavoring agents, or one or moresweetening agents (e.g., sucrose, saccharin). In additional embodiments,dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide dsRNA of this disclosure inadmixture with a dispersing or wetting agent, suspending agent andoptionally one or more preservative, coloring agent, flavoring agent, orsweetening agent.

The present disclosure includes dsRNA compositions prepared for storageor administration that include a pharmaceutically effective amount of adesired compound in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co., A. R. Gennaro edit.,21^(st) Edition, 2005. In certain embodiments, pharmaceuticalcompositions of this disclosure can optionally include preservatives,antioxidants, stabilizers, dyes, flavoring agents, or any combinationthereof. Exemplary preservatives include sodium benzoate, esters ofp-hydroxybenzoic acid, and sorbic acid.

The dsRNA compositions of the instant disclosure can be effectivelyemployed as pharmaceutically-acceptable formulations.Pharmaceutically-acceptable formulations prevent, alter the occurrenceor severity of, or treat (alleviate one or more symptom(s) to adetectable or measurable extent) a disease state or other adversecondition in a subject. A pharmaceutically acceptable formulationincludes salts of the above compounds, for example, acid addition salts,such as salts of hydrochloric acid, hydrobromic acid, acetic acid, orbenzene sulfonic acid. A pharmaceutical composition or formulationrefers to a composition or formulation in a form suitable foradministration into a cell, or a subject such as a human (e.g., systemicadministration). The formulations of the present disclosure, having anamount of dsRNA sufficient to treat or prevent a disorder associatedwith target gene expression are, for example, suitable for topical(e.g., creams, ointments, skin patches, eye drops, ear drops)application or administration. Other routes of administration includeoral, parenteral, sublingual, bladder wash-out, vaginal, rectal,enteric, suppository, nasal, and inhalation. The term parenteral, asused herein, includes subcutaneous, intravenous, intramuscular,intraarterial, intraabdominal, intraperitoneal, intraarticular,intraocular or retrobulbar, intraaural, intrathecal, intracavitary,intracelial, intraspinal, intrapulmonary or transpulmonary,intrasynovial, and intraurethral injection or infusion techniques. Thepharmaceutical compositions of the present disclosure are formulated toallow the dsRNA contained therein to be bioavailable upon administrationto a subject.

In further embodiments, dsRNA of this disclosure can be formulated asoily suspensions or emulsions (e.g., oil-in-water) by suspending dsRNAin, for example, a vegetable oil (e.g., arachis oil, olive oil, sesameoil or coconut oil) or a mineral oil (e.g., liquid paraffin). Suitableemulsifying agents can be naturally-occurring gums (e.g., gum acacia orgum tragacanth), naturally-occurring phosphatides (e.g., soy bean,lecithin, esters or partial esters derived from fatty acids andhexitol), anhydrides (e.g., sorbitan monooleate), or condensationproducts of partial esters with ethylene oxide (e.g., polyoxyethylenesorbitan monooleate). In certain embodiments, the oily suspensions oremulsions can optionally contain a thickening agent, such as beeswax,hard paraffin or cetyl alcohol. In related embodiments, sweeteningagents and flavoring agents can optionally be added to provide palatableoral preparations. In yet other embodiments, these compositions can bepreserved by the optionally adding an anti-oxidant, such as ascorbicacid.

In further embodiments, dsRNA of this disclosure can be formulated assyrups and elixirs with sweetening agents (e.g., glycerol, propyleneglycol, sorbitol, glucose or sucrose). Such formulations can alsocontain a demulcent, preservative, flavoring, coloring agent, or anycombination thereof. In other embodiments, pharmaceutical compositionscomprising dsRNA of this disclosure can be in the form of a sterile,injectable aqueous or oleaginous suspension. The sterile injectablepreparation can also be a sterile, injectable solution or suspension ina non-toxic parenterally acceptable diluent or solvent (e.g., as asolution in 1,3-butanediol). Among the exemplary acceptable vehicles andsolvents useful in the compositions of this disclosure is water,Ringer's solution, or isotonic sodium chloride solution. In addition,sterile, fixed oils may be employed as a solvent or suspending mediumfor the dsRNA of this disclosure. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation of parenteralformulations.

Within certain embodiments of this disclosure, pharmaceuticalcompositions and methods are provided that feature the presence oradministration of one or more dsRNA or analogs thereof of thisdisclosure, combined, complexed, or conjugated with a polypeptide,optionally formulated with a pharmaceutically-acceptable carrier, suchas a diluent, stabilizer, buffer, or the like. The negatively chargeddsRNA molecules of this disclosure may be administered to a patient byany standard means, with or without stabilizers, buffers, or the like,to form a composition suitable for treatment. When it is desired to usea liposome delivery mechanism, standard protocols for formation ofliposomes can be followed. The compositions of the present disclosuremay also be formulated and used as a tablet, capsule or elixir for oraladministration, suppository for rectal administration, sterile solution,or suspension for injectable administration, either with or withoutother compounds known in the art. Thus, dsRNAs of the present disclosuremay be administered in any form, such as nasally, transdermally,parenterally, or by local injection.

In accordance with this disclosure herein, dsRNA molecules (optionallysubstituted or modified or conjugated), compositions thereof, andmethods for inhibiting expression of one or more target gene in a cellor organism are provided. In certain embodiments, this disclosureprovides methods and dsRNA compositions for treating a subject,including a human cell, tissue or individual, having a disease or atrisk of developing a disease caused by or associated with the expressionof one or more target gene. In one embodiment, the method includesadministering a dsRNA of this disclosure or a pharmaceutical compositioncontaining the dsRNA to a cell or an organism, such as a mammal, suchthat expression of the target gene is silenced. Subjects (e.g.,mammalian, human) amendable for treatment using the dsRNA molecules(optionally substituted or modified or conjugated), compositionsthereof, and methods of the present disclosure include those sufferingfrom one or more disease or condition mediated, at least in part, byoverexpression or inappropriate expression of one or more target gene,or which are amenable to treatment by reducing expression of one or moretarget protein, including a hyperproliferative (e.g., cancer),angiogenic, metabolic, or inflammatory (e.g., arthritis) disease ordisorder or condition.

Compositions and methods disclosed herein are useful in the treatment ofa wide variety of target viruses, including retrovirus, such as humanimmunodeficiency virus (HIV), Hepatitis C Virus, Hepatitis B Virus,Coronavirus, as well as respiratory viruses (including human RespiratorySyncytial Virus, human Metapneumovirus, human Parainfluenza virusRhinovirus and Influenza virus.

In other examples, the compositions and methods of this disclosure areuseful as therapeutic tools to regulate expression of one or more targetgene to treat or prevent symptoms of, for example, hyperproliferativedisorders. Exemplary hyperproliferative disorders include neoplasms,carcinomas, sarcomas, tumors, or cancer. More exemplaryhyperproliferative disorders include oral cancer, throat cancer,laryngeal cancer, esophageal cancer, pharyngeal cancer, nasopharyngealcancer, oropharyngeal cancer, gastrointestinal tract cancer,gastrointestinal stromal tumors (GIST), small intestine cancer, coloncancer, rectal cancer, colorectal cancer, anal cancer, pancreaticcancer, breast cancer, cervical cancer, uterine cancer, vulvar cancer,vaginal cancer, urinary tract cancer, bladder cancer, kidney cancer,adrenocortical cancer, islet cell carcinoma, gallbladder cancer, stomachcancer, prostate cancer, ovarian cancer, endometrial cancer,trophoblastic tumor, testicular cancer, penial cancer, bone cancer,osteosarcoma, liver cancer, extrahepatic bile duct cancer, skin cancer,basal cell carcinoma (BCC), lung cancer, small cell lung cancer,non-small cell lung cancer (NSCLC), brain cancer, melanoma, Kaposi'ssarcoma, eye cancer, head and neck cancer, squamous cell carcinoma ofhead and neck, tymoma, thymic carcinoma, thyroid cancer, parathyroidcancer, Hippel-Lindau syndrome, leukemia, acute myeloid leukemia,chronic myelogenous leukemia, acute lymphoblastic leukemia, hairy cellleukemia, lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, T-celllymphoma, multiple myeloma, malignant pleural mesothelioma, Barrett'sadenocarcinoma, Wilm's tumor, or the like.

In other examples, the compositions and methods of this disclosure areuseful as therapeutic tools to regulate expression of one or more targetgene to treat or prevent symptoms of, for example, inflammatorydisorders. Exemplary inflammatory disorders include diabetes mellitus,rheumatoid arthritis, pannus growth in inflamed synovial lining,collagen-induced arthritis, spondylarthritis, ankylosing spondylitis,multiple sclerosis, encephalomyelitis, inflammatory bowel disease,Chron's disease, psoriasis or psoriatic arthritis, myasthenia gravis,systemic lupus erythematosis, graft-versus-host disease,atherosclerosis, and allergies.

Other exemplary disorders that can be treated with dsRNA of the instantdisclosure include metabolic disorders, cardiac disease, pulmonarydisease, neovascularization, ischemic disorders, age-related maculardegeneration, diabetic retinopathy, glomerulonephritis, diabetes,asthma, chronic obstructive pulmonary disease, chronic bronchitis,lymphangiogenesis, and atherosclerosis.

In any of the methods disclosed herein, there may be used one or moredsRNA, or substituted or modified dsRNA as described herein, whichcomprises a first strand that is complementary to a target mRNA and isfully complementary, with up to three mismatches, to at least one otherhuman target family mRNA, or vice-versa, and a second strand and a thirdstrand that is each complementary to non-overlapping regions of thefirst strand, wherein the second strand and third strands can annealwith the first strand to form at least two double-stranded regionsseparated by a gap of up to 10 nucleotides, and wherein at least onedouble-stranded region is from about 5 base pairs up to 13 base pairs.In other embodiments, subjects can be effectively treated,prophylactically or therapeutically, by administering an effectiveamount of one or more dsRNA having a first strand that is complementaryto a target mRNA and is fully complementary, with up to threemismatches, to at least one other target family mRNA, or vice-versa, anda second strand and a third strand that is each complementary tonon-overlapping regions of the first strand, wherein the second strandand third strands can anneal with the first strand to form at least twodouble-stranded regions separated by a gap of up to 10 nucleotides, andwherein at least one double-stranded region is from about 5 base pairsup to 13 base pairs and at least one pyrimidine of the mdRNA issubstituted with a pyrimidine nucleoside according to Formula I or II:

wherein R¹ and R² are each independently a —H, —OH, —OCH₃, —OCH₂OCH₂CH₃,—OCH₂CH₂OCH₃, halogen, substituted or unsubstituted C₁-C₁₀ alkyl,alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino,aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl,trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted —O-allyl,—O—CH₂CH═CH₂, —O—CH═CHCH₃, substituted or unsubstituted C₂-C₁₀ alkynyl,carbamoyl, carbamyl, carboxy, carbonylamino, substituted orunsubstituted aryl, substituted or unsubstituted aralkyl, —NH₂, —NO₂,—C≡N, or heterocyclo group; R³ and R⁴ are each independently a hydroxyl,a protected hydroxyl, or an internucleoside linking group; and R⁵ and R⁸are independently O or S. In certain embodiments, at least onenucleoside is according to Formula I in which R¹ is methyl and R² is—OH, or R¹ is methyl, R² is —OH, and R⁸ is S. In other embodiments, theinternucleoside linking group covalently links from about 5 to about 40nucleosides.

In further embodiments, subjects can be effectively treated,prophylactically or therapeutically, by administering an effectiveamount of one or more dsRNA, or substituted or modified dsRNA asdescribed herein, having a first strand that is complementary to atarget mRNA and is fully complementary, with up to three mismatches, toat least one other target gene mRNA, or vice-versa, and a second strandand a third strand that is each complementary to non-overlapping regionsof the first strand, wherein the second strand and third strands cananneal with the first strand to form at least two double-strandedregions separated by a gap of up to 10 nucleotides, and wherein thecombined double-stranded regions total about 15 base pairs to about 40base pairs and the mdRNA molecule comprises blunt ends. In still furtherembodiments, methods disclosed herein there may be used with one or moredsRNA that comprises a first strand that is complementary to a targetgene mRNA and is fully complementary, with up to three mismatches, to atleast one other target family mRNA, or vice-versa, and a second strandand a third strand that is each complementary to non-overlapping regionsof the first strand, wherein the second strand and third strands cananneal with the first strand to form at least two double-strandedregions separated by a gap of up to 10 nucleotides, and wherein at leastone double-stranded region is from about 5 base pairs up to 13 basepairs, the mdRNA molecule comprises blunt ends, and at least onepyrimidine of the mdRNA is substituted with a pyrimidine nucleosideaccording to Formula I or II:

wherein R¹ and R² are each independently a —H, —OH, —OCH₃, —OCH₂OCH₂CH₃,—OCH₂CH₂OCH₃, halogen, substituted or unsubstituted C₁-C₁₀ alkyl,alkoxy, alkoxyalkyl, hydroxyalkyl, carboxyalkyl, alkylsulfonylamino,aminoalkyl, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, haloalkyl,trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted —O-allyl,—O—CH₂CH═CH₂, —O—CH═CHCH₃, substituted or unsubstituted C₂-C₁₀ alkynyl,carbamoyl, carbamyl, carboxy, carbonylamino, substituted orunsubstituted aryl, substituted or unsubstituted aralkyl, —NH₂, —NO₂,—C≡N, or heterocyclo group; R³ and R⁴ are each independently a hydroxyl,a protected hydroxyl, or an internucleoside linking group; and R⁵ and R⁸are independently O or S. In certain embodiments, at least onenucleoside is according to Formula I in which R¹ is methyl and R² is—OH, or R¹ is methyl, R² is —OH, and R⁸ is S. In other embodiments, theinternucleoside linking group covalently links from about 5 to about 40nucleosides.

Within additional aspects of this disclosure, combination formulationsand methods are provided comprising an effective amount of one or moredsRNA of the present disclosure in combination with one or moresecondary or adjunctive active agents that are formulated together oradministered coordinately with the dsRNA of this disclosure to controlone or more target gene-associated disease or condition as describedherein. Useful adjunctive therapeutic agents in these combinatorialformulations and coordinate treatment methods include, for example,enzymatic nucleic acid molecules, allosteric nucleic acid molecules,antisense, decoy, or aptamer nucleic acid molecules, antibodies such asmonoclonal antibodies, small molecules and other organic or inorganiccompounds including metals, salts and ions, and other drugs and activeagents indicated for treating one or more target gene-associated diseaseor condition, including chemotherapeutic agents used to treat cancer,steroids, non-steroidal anti-inflammatory drugs (NSAIDs), or the like.

Exemplary chemotherapeutic agents include alkylating agents (e.g.,cisplatin, oxaliplatin, carboplatin, busulfan, nitrosoureas, nitrogenmustards, uramustine, temozolomide), antimetabolites (e.g., aminopterin,methotrexate, mercaptopurine, fluorouracil, cytarabine), taxanes (e.g.,paclitaxel, docetaxel), anthracyclines (e.g., doxorubicin, daunorubicin,epirubicin, idaruicin, mitoxantrone, valrubicin), bleomycin, mytomycin,actinomycin, hydroxyurea, topoisomerase inhibitors (e.g., camptothecin,topotecan, irinotecan, etoposide, teniposide), monoclonal antibodies(e.g., alemtuzumab, bevacizumab, cetuximab, gemtuzumab, panitumumab,rituximab, tositumomab, trastuzumab), vinca alkaloids (e.g.,vincristine, vinblastine, vindesine, vinorelbine), cyclophosphamide,prednisone, leucovorin, oxaliplatin.

To practice the coordinate administration methods of this disclosure, adsRNA is administered simultaneously or sequentially in a coordinatedtreatment protocol with one or more secondary or adjunctive therapeuticagents described herein or known in the art. The coordinateadministration may be done in either order, and there may be a timeperiod while only one or both (or all) active therapeutic agents,individually or collectively, exert their biological activities. Adistinguishing aspect of all such coordinate treatment methods is thatthe dsRNA present in a composition elicits some favorable clinicalresponse, which may or may not be in conjunction with a secondaryclinical response provided by the secondary therapeutic agent. Forexample, the coordinate administration of the dsRNA with a secondarytherapeutic agent as contemplated herein can yield an enhanced (e.g.,synergistic) therapeutic response beyond the therapeutic responseelicited by either or both the purified dsRNA or secondary therapeuticagent alone.

In another embodiment, a dsRNA of this disclosure can include aconjugate member on one or more of the nucleotides of a dsRNA (e.g.,terminal). The conjugate member can be, for example, a lipophile, aterpene, a protein binding agent, a vitamin, a carbohydrate, or apeptide. For example, the conjugate member can be naproxen, nitroindole(or another conjugate that contributes to stacking interactions),folate, ibuprofen, or a C5 pyrimidine linker. In other embodiments, theconjugate member is a glyceride lipid conjugate (e.g., a dialkylglyceride derivatives), vitamin E conjugates, or thio-cholesterols.Additional conjugate members include peptides that function, whenconjugated to a modified dsRNA of this disclosure, to facilitatesdelivery of the dsRNA into a target cell, or otherwise enhance delivery,stability, or activity of the dsRNA when contacted with a biologicalsample. Exemplary peptide conjugate members for use within these aspectsof this disclosure, include peptides PN27, PN28, PN29, PN58, PN61, PN73,PN158, PN159, PN173, PN182, PN202, PN204, PN250, PN361, PN365, PN404,PN453, and PN509 are described, for example, in U.S. Patent ApplicationPublication Nos. 2006/0040882 and 2006/0014289, and U.S. ProvisionalPatent Application No. 60/939,578, which are all incorporated herein byreference. In certain embodiments, when peptide conjugate partners areused to enhance delivery of dsRNA or analogs thereof of this disclosure,the resulting dsRNA formulations and methods will often exhibit furtherreduction of an interferon response in target cells as compared todsRNAs delivered in combination with alternate delivery vehicles, suchas lipid delivery vehicles (e.g., Lipofectamine™).

In still another embodiment, a dsRNA or analog thereof of thisdisclosure may be conjugated to the polypeptide and admixed with one ormore non-cationic lipids or a combination of a non-cationic lipid and acationic lipid to form a composition that enhances intracellulardelivery of the dsRNA as compared to delivery resulting from contactingthe target cells with a naked dsRNA. In more detailed aspects of thisdisclosure, the mixture, complex or conjugate comprising a dsRNA and apolypeptide can be optionally combined with (e.g., admixed or complexedwith) a cationic lipid, such as Lipofectine™. To produce thesecompositions comprised of a polypeptide, dsRNA and a cationic lipid, thedsRNA and peptide may be mixed together first in a suitable medium suchas a cell culture medium, after which the cationic lipid is added to themixture to form a dsRNA/delivery peptide/cationic lipid composition.Optionally, the peptide and cationic lipid can be mixed together firstin a suitable medium such as a cell culture medium, followed by theaddition of the dsRNA to form the dsRNA/delivery peptide/cationic lipidcomposition.

This disclosure also features the use of dsRNA compositions comprisingsurface-modified liposomes containing poly(ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues (Lasic et al., Chem. Rev. 95:2601, 1995;Ishiwata et al., Chem. Pharm. Bull. 43:1005, 1995). Such liposomes havebeen shown to accumulate selectively in tumors, presumably byextravasation and capture in the neovascularized target tissues (Lasicet al., Science 267:1275, 1995; Oku et al., Biochim. Biophys. Acta1238:86, 1995). The long-circulating liposomes enhance thepharmacokinetics and pharmacodynamics of nucleic acid molecules ascompared to conventional cationic liposomes, which are known toaccumulate in tissues of the mononuclear phagocytic system (MPS) (Liu etal., J. Biol. Chem. 42:24864, 1995; Choi et al., PCT Publication No. WO96/10391; Ansell et al., PCT Publication No. WO 96/10390; Holland etal., PCT Publication No. WO 96/10392). Long-circulating liposomes mayalso provide additional protection from nuclease degradation as comparedto cationic liposomes in theory due to avoiding accumulation inmetabolically aggressive MPS tissues, such as the liver and spleen.

In one embodiment, this disclosure provides compositions suitable foradministering dsRNA molecules of this disclosure to specific cell types,such as hepatocytes. For example, the asialoglycoprotein receptor(ASGPr) (Wu and Wu, J. Biol. Chem. 262:4429, 1987) is unique tohepatocytes and binds branched galactose-terminal glycoproteins, such asasialoorosomucoid (ASOR). Binding of such glycoproteins or syntheticglycoconjugates to the receptor takes place with an affinity thatstrongly depends on the degree of branching of the oligosaccharidechain, for example, triatennary structures are bound with greateraffinity than biatenarry or monoatennary chains (Baenziger and Fiete,Cell 22: 611, 1980; Connolly et al., J. Biol. Chem. 257:939, 1982). Leeand Lee (Glycoconjugate J. 4:317, 1987) obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., J. Med. Chem. 24:1388, 1981). The use of galactose andgalactosamine based conjugates to transport exogenous compounds acrosscell membranes can provide a targeted delivery approach to the treatmentof liver disease. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use of dsRNAbioconjugates of this disclosure.

The present disclosure also features a method for preparing dsRNAnanoparticles. A first solution containing melamine derivatives isdissolved in an organic solvent such as dimethyl sulfoxide, or dimethylformamide to which an acid such as HCl has been added. The concentrationof HCl would be about 3.3 moles of HCl for every mole of the melaminederivative. The first solution is then mixed with a second solution,which includes a nucleic acid dissolved or suspended in a polar orhydrophilic solvent (e.g., an aqueous buffer solution containing, forinstance, ethylenediaminetraacetic acid (EDTA), or tris(hydroxymethyl)aminomethane (TRIS), or combinations thereof. The mixture forms a firstemulsion. The mixing can be done using any standard technique such as,for example, sonication, vortexing, or in a microfluidizer. This causescomplexing of the nucleic acids with the melamine derivative forming atrimeric nucleic acid complex. While not being bound to theory ormechanism, it is believed that three nucleic acids are complexed in acircular fashion about one melamine derivative moiety, and that a numberof the melamine derivative moieties can be complexed with the threenucleic acid molecules depending on the size of the number ofnucleotides that the nucleic acid has. The concentration should be fromabout 1 to about 7 moles of the melamine derivative for every mole of adouble-stranded nucleic acid having about 20 nucleotide pairs, more ifthe double-stranded nucleic acid is larger. The resultant nucleic acidparticles can be purified and the organic solvent removed usingsize-exclusion chromatography or dialysis or both.

The complexed nucleic acid nanoparticles can then be mixed with anaqueous solution containing either polyarginine or a Gln-Asn polymer, orboth, in an aqueous solution. A preferred molecular weight of eachpolymer is about 5000 to about 15,000 Daltons. This forms a solutioncontaining nanoparticles of nucleic acid complexed with the melaminederivative and the polyarginine and the Gln-Asn polymers. The mixingsteps are carried out in a manner that minimizes shearing of the nucleicacid while producing nanoparticles on average smaller than about 200nanometers in diameter. While not wishing to be bound by theory, it isbelieved that the polyarginine complexes with the negative charge of thephosphate groups within the minor groove of the nucleic acid, and thepolyarginine wraps around the trimeric nucleic acid complex. At eitherterminus of the polyarginine other moieties, such as the TATpolypeptide, mannose or galactose, can be covalently bound to thepolymer to direct binding of the nucleic acid complex to specifictissues, such as to the liver when galactose is used. While not beingbound to theory, it is believed that the Gln-Asn polymer complexes withthe nucleic acid complex within the major groove of the nucleic acidthrough hydrogen bonding with the bases of the nucleic acid. Thepolyarginine and the Gln-Asn polymer should be present at aconcentration of 2 moles per every mole of nucleic acid having 20 basepairs. The concentration should be increased proportionally for anucleic acid having more than 20 base pairs. For example, if the nucleicacid has 25 base pairs, the concentration of the polymers should be2.5-3 moles per mole of double-stranded nucleic acid. An example of is apolypeptide operatively linked to an N-terminal protein transductiondomain from HIV TAT. The HIV TAT construct for use in such a protein isdescribed in detail in Vocero-Akbani et al., Nature Med. 5:23, 1999. Seealso, U.S. Patent Application Publication No. 2004/0132161, published onJul. 8, 2004. The resultant nanoparticles can be purified by standardmeans such as size exclusion chromatography followed by dialysis. Thepurified complexed nanoparticles can then be lyophilized usingtechniques well known in the art.

One embodiment of the present disclosure provides nanoparticles lessthan 100 nanometers (nm) comprising dsRNA that decreases expression ofone or more target gene by RNAi. More specifically, the dsRNA is lessthan about 30 base pairs in length, or is from about 20 to about 25 basepairs in length.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of subject being treated, the physicalcharacteristics of the specific subject under consideration fortreatment, concurrent medication, and other factors that those skilledin the medical arts will recognize. For example, an amount between 0.1mg/kg and 100 mg/kg body weight/day of active ingredients may beadministered depending on the potency of a dsRNA of this disclosure.

Dosage levels in the order of about 0.1 mg to about 140 mg per kilogramof body weight per day can be useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per patient perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular patientdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy. Following administration of dsRNA according to the formulationsand methods of this disclosure, test subjects will exhibit about a 10%up to about a 99% reduction in one or more symptoms associated with thedisease or disorder being treated, as compared to placebo-treated orother suitable control subjects.

Nucleic acid molecules and polypeptides can be administered to cells bya variety of methods known to those of skill in the art, includingadministration within formulations that comprise the dsRNA andpolypeptide alone, or that further comprise one or more additionalcomponents, such as a pharmaceutically acceptable carrier, diluent,excipient, adjuvant, emulsifier, buffer, stabilizer, preservative, orthe like. In certain embodiments, the dsRNA or the polypeptide can beencapsulated in liposomes, administered by iontophoresis, orincorporated into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, bioadhesive microspheres, or proteinaceousvectors (see, e.g., PCT Publication No. WO 00/53722). Alternatively, anucleic acid/peptide/vehicle combination can be locally delivered bydirect injection or by use of an infusion pump. Direct injection of thenucleic acid molecules of this disclosure, whether subcutaneous,intramuscular, or intradermal, can take place using standard needle andsyringe methodologies, or by needle-free technologies, such as thosedescribed in Conroy et al., Clin. Cancer Res. 5:2330, 1999, and PCTPublication No. WO 99/31262.

The dsRNAs can also be administered in the form of suppositories, forexample, for rectal administration of the drug. These compositions canbe prepared by mixing the drug with a suitable non-irritating excipientthat is solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum to release the drug.Such materials include cocoa butter and polyethylene glycols.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

Further methods for delivery of nucleic acid molecules, such as thedsRNAs of this disclosure, are described, for example, in Boado et al.,J. Pharm. Sci. 87:1308, 1998; Tyler et al., FEBS Lett. 421:280, 1999;Pardridge et al., Proc. Nat'l Acad. Sci. USA 92:5592, 1995; Boado, Adv.Drug Delivery Rev. 15:73, 1995; Aldrian-Herrada et al., Nucleic AcidsRes. 26:4910, 1998; Tyler et al., Proc. Nat'l Acad. Sci. USA 96:7053,1999; Akhtar et al., Trends Cell Bio. 2:139, 1992; “Delivery Strategiesfor Antisense Oligonucleotide Therapeutics,” ed. Akhtar, 1995, Maurer etal., Mol. Membr. Biol. 16:129, 1999; Hofland and Huang, Handb. Exp.Pharmacol. 137:165, 1999; and Lee et al., ACS Symp. Ser. 752:184, 2000.Sullivan et al. (PCT Publication No. WO 94/02595) further describegeneral methods for delivery of enzymatic nucleic acid molecules, whichmethods can be used to supplement or complement delivery of dsRNAcontemplated within this disclosure.

In addition to in vivo gene inhibition, a skilled artisan willappreciate that the dsRNAs of the present disclosure are useful in awide variety of in vitro applications, such as in scientific andcommercial research (e.g., elucidation of physiological pathways, drugdiscovery and development), and medical and veterinary diagnostics. Ingeneral, the method involves the introduction of the dsRNA agent into acell using known techniques (e.g., absorption through cellularprocesses, or by auxiliary agents or devices, such as electroporation,lipofection, or through the use of peptide conjugates), then maintainingthe cell for a time sufficient to obtain degradation of one or moretarget mRNA.

All U.S. patents, U.S. patent application publications, U.S. patentapplications, foreign patents, foreign patent applications, non-patentpublications, figures, and websites referred to in this specificationare expressly incorporated herein by reference, in their entirety.

EXAMPLES Example 1 Knockdown of β-Galactosidase Activity by Gapped dsRNADicer Substrate

The activity of a Dicer substrate dsRNA containing a gap in thedouble-stranded structure in silencing LacZ mRNA as compared to thenormal Dicer substrate dsRNA (i.e., not having a gap) was examined.

Nucleotide Sequences of dsRNA and mdRNA Targeting LacZ mRNA

The nucleic acid sequence of the one or more sense strands, and theantisense strand of the dsRNA and gapped dsRNA (also referred to hereinas a meroduplex or mdRNA) are shown below and were synthesized usingstandard techniques. The RISC activator LacZ dsRNA comprises a 21nucleotide sense strand and a 21 nucleotide antisense strand, which cananneal to form a double-stranded region of 19 base pairs with a twodeoxythymidine overhang on each strand (referred to as 21/21 dsRNA).

LacZ dsRNA (21/21)—RISC Activator

(SEQ ID NO: 1) Sense 5′-CUACACAAAUCAGCGAUUUdTdT-3′ (SEQ ID NO: 2)Antisense 3′-dTdTGAUGUGUUUAGUCGCUAAA-5′

The Dicer substrate LacZ dsRNA comprises a 25 nucleotide sense strandand a 27 nucleotide antisense strand, which can anneal to form adouble-stranded region of 25 base pairs with one blunt end and acytidine and uridine overhang on the other end (referred to as 25/27dsRNA).

LacZ dsRNA (25/27)—Dicer Substrate

(SEQ ID NO: 3) Sense 5′-CUACACAAAUCAGCGAUUUCCAUdGdT-3′ (SEQ ID NO: 4)Antisense 3′-CUGAUGUGUUUAGUCGCUAAAGGUA C A- 5′

The LacZ mdRNA comprises two sense strands of 13 nucleotides(5′-portion) and 11 nucleotides (3′-portion) and a 27 nucleotideantisense strand, which three strands can anneal to form twodouble-stranded regions of 13 and 11 base pairs separated by a singlenucleotide gap (referred to as a 13, 11/27 mdRNA). The 5′-end of the 11nucleotide sense strand fragment may be optionally phosphorylated. The“*” indicates a gap—in this case, a single nucleotide gap (i.e., acytidine is missing).

LacZ mdRNA (13, 11/27)—Dicer Substrate

(SEQ ID NOS: 5, 6) Sense 5′-CUACACAAAUCAG*GAUUUCCAUdGdT-3′(SEQ ID NO: 4) Antisense 3′-CUGAUGUGUUUAGUCGCUAAAGGUA C A- 5′Each of the LacZ dsRNA or mdRNA was used to transfect 9lacZ/R cells.

Transfection

Six well collagen-coated plates were seeded with 5×10⁵ 9lacZ/Rcells/well in a 2 ml volume per well, and incubated overnight at 37°C./5% CO₂ in DMEM/high glucose media. Preparation for transfection: 250μl of OPTIMEM media without serum was mixed with 5 μl of 20 pmol/μldsRNA and 5 μl of HIPERFECT transfection solution (Qiagen) was mixedwith another 250 μl OPTIMEM media. After both mixtures were allowed toequilibrate for 5 minutes, the RNA and transfection solutions werecombined and left at room temperature for 20 minutes to formtransfection complexes. The final concentration of HIPERFECT was 50 μM,and the dsRNAs were tested at 0.05 nM, 0.1 nM, 0.2 nM, 0.5 nM, 1 nM, 2nM, 5 nM, and 10 nM, while the mdRNA was tested at 0.2 nM, 0.5 nM, 1 nM,2 nM, 5 nM, 10 nM, 20 nM, and 50 nM. Complete media was removed, thecells were washed with incomplete OPTIMEM, and then 500 transfectionmixture was applied to the cells, which were incubated with gentleshaking at 37° C. for 4 hours. After transfecting, the transfectionmedia was removed, cells were washed once with complete DMEM/highglucose media, fresh media added, and the cells were then incubated for48 hours at 37° C., 5% CO₂.

β-Galactosidase Assay

Transfected cells were washed with PBS, and then detached with 0.5 mltrypsin/EDTA. The detached cells were suspended in 1 ml completeDMEM/high glucose and transferred to a clean tube. The cells wereharvested by centrifugation at 250×g for 5 minutes, and then resuspendedin 50 μl 1× lysis buffer at 4° C. The lysed cells were subjected to twofreeze-thaw cycles on dry ice and a 37° C. water bath. The lysed sampleswere centrifuged for 5 minutes at 4° C. and the supernatant wasrecovered. For each sample, 1.5 μl and 10 μl of lysate was transferredto a clean tube and sterile water added to a final volume of 30 μlfollowed by the addition of 70 μl o-nitrophenyl-β-D-galactopyranose(ONPG) and 200 μl 1× cleavage buffer with β-mercaptoethanol. The sampleswere mixed briefly, incubated for 30 minutes at 37° C., and then 500 μlstop buffer was added (final volume 800 μl). β-Galactosidase activityfor each sample was measured in disposable cuvettes at 420 nm. Proteinconcentration was determined by the BCA (bicinchoninic acid) method. Forthe purpose of the instant example, the level of measured LacZ activitywas correlated with the quantity of LacZ transcript within 9L/LacZcells. Thus, a reduction in β-galactosidase activity after dsRNAtransfection, absent a negative impact on cell viability, was attributedto a reduction in the quantity of LacZ transcripts resulting fromtargeted degradation mediated by the LacZ dsRNA.

Results

Knockdown activity in transfected and untransfected cells was normalizedto a Qneg control dsRNA and presented as a normalized value of the Qnegcontrol (i.e., Qneg represented 100% or “normal” gene expressionlevels). Both the lacZ RISC activator and Dicer substrate dsRNAsmolecule showed good knockdown of β-galactosidase activity atconcentration as low as 0.1 nM (FIG. 1), while the Dicer substrateantisense strand alone (single stranded 27mer) had no silencing effect.Surprisingly, a gapped mdRNA showed good knockdown although somewhatlower than that of intact RISC activator and Dicer substrate dsRNAs(FIG. 1). The presence of the gapmer cytidine (i.e., the missingnucleotide) at various concentrations (0.1 μM to 50 μM) had no effect onthe activity of the mdRNA (data not shown). None of the dsRNA or mdRNAsolutions showed any detectable toxicity in the transfected 9L/LacZcells. The IC₅₀ of the lacZ mdRNA was calculated to be 3.74 nM, which isabout 10 fold lower than what had been previously measured for lacZdsRNA 21/21 (data not shown). These results show that a meroduplex(gapped dsRNA) is capable of inducing gene silencing.

Example 2 Knockdown of Influenza Gene Expression by Nicked dsRNA

The activity of a nicked dsRNA (21/21) in silencing influenza geneexpression as compared to a normal dsRNA (i.e., not having a nick) wasexamined.

Nucleotide Sequences of dsRNA and mdRNA Targeting Influenza mRNA

The dsRNA and nicked dsRNA (another form of meroduplex, referred toherein as ndsRNA) are shown below and were synthesized using standardtechniques. The RISC activator influenza G1498 dsRNA comprises a 21nucleotide sense strand and a 21 nucleotide antisense strand, which cananneal to form a double-stranded region of 19 base pairs with a twodeoxythymidine overhang on each strand.

G1498-wt dsRNA (21/21)

(SEQ ID NO: 7) Sense 5′-GGAUCUUAUUUCUUCGGAGdTdT-3′ (SEQ ID NO: 8)Antisense 3′-dTdTCCUAGAAUAAAGAAGCCUC-5′

The RISC activator influenza G1498 dsRNA was nicked on the sense strandafter nucleotide 11 to produce a ndsRNA having two sense strands of 11nucleotides (5′-portion, italic) and 10 nucleotides (3′-portion) and a21 nucleotide antisense strand, which three strands can anneal to formtwo double-stranded regions of 11 (shown in italics) and 10 base pairsseparated by a one nucleotide gap (which may be referred to as G1498 11,10/21 ndsRNA-wt). The 5′-end of the 10 nucleotide sense strand fragmentmay be optionally phosphorylated, as depicted by a “p” preceding thenucleotide (e.g., pC).

G1498 ndsRNA-wt (11, 10/21)

(SEQ ID NO: 9, 10) Sense 5′-GGAUCUUAUUUCUUCGGAGdTdT-3′ (SEQ ID NO: 8)Antisense 3′-dTdTCCUAGAAUAAAGAAGCCUC-5′G1498 ndsRNA-wt (11, 10/21)

(SEQ ID NOS: 9, 10) Sense 5′-GGAUCUUAUUUpCUUCGGAGdTdT-3′ (SEQ ID NO: 8)Antisense 3′-dTdTCCUAGAAUAAAGAAGCCUC-5′In addition, each of these G1498 dsRNAs were made with each Usubstituted with a 5-methyluridine (ribothymidine) and are referred toas G1498 dsRNA-rT. Each of the G1498 dsRNA or ndsRNA (meroduplex), withor without the 5-methyluridine substitution, was used to transfect HeLaS3 cells having an influenza target sequence associated with aluciferase gene. Also, the G1498 antisense strand alone or the antisensestrand annealed to the 11 nucleotide sense strand portion alone or the10 nucleotide sense strand portion alone were examined for activity.

Transfection and Dual Luciferase Assay

The reporter plasmid psiCHECK™-2 (Promega, Madison, Wis.), whichconstitutively expresses both firefly luc2 (Photinus pyralis) andRenilla (Renilla reniformis, also known as sea pansy) luciferases, wasused to clone in a portion of the influenza NP gene downstream of theRenilla translational stop codon that results in a Renilla-influenza NPfusion mRNA. The firefly luciferase in the psiCHECK™-2 vector is used tonormalize Renilla luciferase expression and serves as a control fortransfection efficiency.

Multi-well plates were seeded with HeLa S3 cells/well in 100 μl Ham'sF12 medium and 10% fetal bovine serum, and incubated overnight at 37°C./5% CO₂. Using essentially the same transfection procedure asdescribed in Example 1, the HeLa S3 cells were transfected with thepsiCHECK™-influenza plasmid (75 ng) and G1498 dsRNA or ndsRNA (finalconcentration of 10 nM or 100 nM) formulated in Lipofectamine™ 2000 andOPTIMEM reduced serum medium. The transfection mixture was incubatedwith the HeLa S3 cells with gentle shaking at 37° C. for about 18 to 20hours.

After transfecting, firefly luciferase reporter activity was measuredfirst by adding Dual-G10™ Luciferase Reagent (Promega, Madison, Wis.)for 10 minutes with shaking, and then quantitating the luminescentsignal using a VICTOR³™ 1420 Multilabel Counter (PerkinElmer, Waltham,Mass.). After measuring the firefly luminescence, Stop & Glo® Reagent(Promega, Madison, Wis.) was added for 10 minutes with shaking tosimultaneously quench the firefly reaction and initiate the Renillaluciferase reaction, and then the Renilla luciferase luminescent signalwas quantitated VICTOR³™ 1420 Multilabel Counter (PerkinElmer, Waltham,Mass.).

Results

Knockdown activity in transfected and untransfected cells was normalizedto a Qneg control dsRNA and presented as a normalized value of the Qnegcontrol (i.e., Qneg represented 100% or “normal” gene expressionlevels). Thus, a smaller value indicates a greater knockdown effect. TheG1498 dsRNA-wt and dsRNA-rT showed similar good knockdown at a 100 nMconcentration (FIG. 2). Surprisingly, the G1498 ndsRNA-rT, whetherphosphorylated or not, showed good knockdown although somewhat lowerthan the G1498 dsRNA-wt (FIG. 2). Similar results were obtained withdsRNA or ndsRNA at 10 nM (data not shown). None of the G1498 dsRNA orndsRNA solutions showed any detectable toxicity in HeLa S3 cells ateither 10 nM or 100 nM. Even the presence of only half a nicked sensestrand (an 11 nucleotide or 10 nucleotide strand alone) with a G1498antisense strand showed some detectable activity. These results showthat a nicked-type meroduplex dsRNA molecule is unexpectedly capable ofpromoting gene silencing.

Example 3 Knockdown Activity of mdRNA Having a Nick in DifferentPositions

In this example, the activity of a dicer substrate LacZ dsRNA of Example1 having a sense strand with a nick at various positions was examined.In addition, a dideoxy nucleotide (i.e., ddG) was incorporated at the5′-end of the 3′-most strand of a sense sequence having a nick or asingle nucleotide gap to determine whether the in vivo ligation of thenicked sense strand is “rescuing” activity. The ddG is not a substratefor ligation. Also examined was the influenza dicer substrate dsRNA ofExample 6 having a sense strand with a nick at one of positions 8 to 14.The “p” designation indicates that the 5′-end of the 3′-most strand ofthe nicked sense influenza sequence was phosphorylated. The “L”designation indicates that the G at position 2 of the 5′-most strand ofthe nicked sense influenza sequence was substituted for a locked nucleicacid G. The Qneg is a negative control dsRNA.

The dual fluorescence assay of Example 2 was used to measure knockdownactivity with 5 nM of the LacZ sequences and 0.5 nM of the influenzasequences. The lacZ dicer substrate (25/27, LacZ-DS) and lacZ RISCactivator (21/21, LacZ) are equally active, and the LacZ-DS can benicked in any position between 8 and 14 without affecting activity (FIG.3). In addition, the inclusion of a ddG on the 5′-end of the 3′-mostLacZ sense sequence having a nick (LacZ:DSNkd13-3′dd) or a onenucleotide gap (LacZ:DSNkd13D1-3′dd) was essentially as active as theunsubstituted sequence (FIG. 3). The influenza dicer substrate (G1498DS)nicked at any one of positions 8 to 14 was also highly active (FIG. 4).Phosphorylation of the 5′-end of the 3′-most strand of the nicked senseinfluenza sequence had essentially no effect on activity, but additionof a locked nucleic acid appears to improve activity.

Example 4 Mean Inhibitory Concentration of mdRNA

In this example, a dose response assay was performed to measure the meaninhibitory concentration (IC₅₀) of the influenza dicer substrate dsRNAof Example 7 having a sense strand with a nick at position 12, 13, or14, including or not a locked nucleic acid. The dual luciferase assay ofExample 2 was used. The influenza dicer substrate dsRNA (G1498DS) wastested at 0.0004 nM, 0.002 nM, 0.005 nM, 0.019 nM, 0.067 nM, 0.233 nM,0.816 nM, 2.8 nM, and 10 nM, while the mdRNA with a nick at position 13(G1498DS:Nkd13) was tested at 0.001 nM, 0.048 nM, 0.167 nM, 1 nM, 2 nM,7 nM, and 25 nM (see FIG. 5). Also tested were RISC activator molecules(21/21) with or without a nick at various positions, G1498DS:Nkd12, andG1498DS:Nkd14, each of the nicked versions with a locked nucleic acid asdescribed above (data not shown). The Qneg is a negative control dsRNA.

The IC₅₀ of the RISC activator G1498 was calculated to be about 22 pM,while the dicer substrate G1498DS IC₅₀ was calculated to be about 6 pM.The IC₅₀ of RISC and Dicer mdRNAs range from about 200 pM to about 15nM. The inclusion of a single locked nucleic acid reduced the IC₅₀ ofDicer mdRNAs by up 4 fold (data not shown). These results show that ameroduplex dsRNA having a nick or gap in any position is capable ofinducing gene silencing.

Example 5 Knockdown Activity of mdRNA Having a Gap of Different Sizesand Positions

The activity of an influenza dicer substrate dsRNA having a sense strandwith a gap of differing sizes and positions was examined. The influenzadicer substrate dsRNA of Example 7 was generated with a sense strandhaving a gap of 0 to 6 nucleotides at position 8, a gap of 4 nucleotidesat position 9, a gap of 3 nucleotides at position 10, a gap of 2nucleotides at position 11, and a gap of 1 nucleotide at position 12(see Table 3). The Qneg is a negative control dsRNA. Each of the mdRNAswere tested at a concentration of 5 nM (data not shown) and 10 nM. ThemdRNAs have the following antisense strand5′-CAUUGUCUCCGAAGAAAUAAGAUCCUU (SEQ ID NO:11) and nicked or gapped sensestrands as shown in Table 3.

TABLE 3 5′ Sense* 3′ Sense Gap Gap % mdRNA (SEQ ID NO.) (SEQ ID NO.) PosSize KD G1498:DSNkd8 G G AUCUUA (12) UUUCUUCGGAGACAAdTdG (13)  8 0 67.8G1498:DSNkd8D1 G G AUCUUA (12)  UUCUUCGGAGACAAdTdG (14)  8 1 60.9G1498:DSNkd8D2 G G AUCUUA (12)   UCUUCGGAGACAAdTdG (15)  8 2 48.2G1498:DSNkd8D3 G G AUCUUA (12)    CUUCGGAGACAAdTdG (16)  8 3 44.1G1498:DSNkd8D4 G G AUCUUA (12)     UUCGGAGACAAdTdG (17)  8 4 30.8G1498:DSNkd8D5 G G AUCUUA (12)      UCGGAGACAAdTdG (18)  8 5 10.8G1498:DSNkd8D6 G G AUCUUA (12)       CGGAGACAAdTdG (19)  8 6 17.9G1498:DSNkd9D4 G G AUCUUAU (20)      UCGGAGACAAdTdG (18)  9 4 38.9G1498:DSNkd10D3 G G AUCUUAUU (21)      UCGGAGACAAdTdG (18) 10 3 38.4G1498:DSNkd11D2 G G AUCUUAUUU (22)      UCGGAGACAAdTdG (18) 11 2 46.2G1498:DSNkd12D1 G G AUCUUAUUUC (23)      UCGGAGACAAdTdG (18) 12 1 49.6Plasmid — — — — 5.3 * G indicates a locked nucleic acid G in the 5′sense strand.

The dual fluorescence assay of Example 2 was used to measure knockdownactivity. Similar results were obtained at both the 5 nM and 10 nMconcentrations. These data show that an mdRNA having a gap of up to 6nucleotides still has activity, although having four or fewer missingnucleotides shows the best activity (see, also, FIG. 6). Thus, mdRNAhaving various sizes gaps that are in various different positions haveknockdown activity.

To examine the general applicability of a sequence having a sense strandwith a gap of differing sizes and positions, a different dsRNA sequencewas tested. The lacZ RISC dsRNA of Example 1 was generated with a sensestrand having a gap of 0 to 6 nucleotides at position 8, a gap of 5nucleotides at position 9, a gap of 4 nucleotides at position 10, a gapof 3 nucleotides at position 11, a gap of 2 nucleotides at position 12,a gap of 1 nucleotide at position 12, and a nick (gap of 0) at position14 (see Table 4). The Qneg is a negative control dsRNA. Each of themdRNAs was tested at a concentration of 5 nM (data not shown) and 25 nM.The lacZ mdRNAs have the following antisense strand5′-AAAUCGCUGAUUUGUGUAGdTdTUAAA (SEQ ID NO:2) and nicked or gapped sensestrands as shown in Table 4.

TABLE 4 5′ Sense* 3′ Sense* Gap Gap mdRNA (SEQ ID NO.) (SEQ ID NO.) PosSize LacZ:Nkd8 CU A CACAA (24) AUCAGCG A UUUdTdT (25)  8 0 LacZ:Nkd8D1CU A CACAA (24)  UCAGCG A UUUdTdT (26)  8 1 LacZ:Nkd8D2 CU A CACAA (24)  CAGCG A UUUdTdT (27)  8 2 LacZ:Nkd8D3 CU A CACAA (24)    AGCG AUUUdTdT (28)  8 3 LacZ:Nkd8D4 CU A CACAA (24)     GCG A UUUdTdT (29)  84 LacZ:Nkd8D5 CU A CACAA (24)      CG A UUUdTdT (30)  8 5 LacZ:Nkd8D6 CUA CACAA (24)       G A UUUdTdT (31)  8 6 LacZ:Nkd9D5 CU A CACAAA (32)      G A UUUdTdT (31)  9 5 LacZ:Nkd10D4 CU A CACAAAU (33)       G AUUUdTdT (31) 10 4 LacZ:Nkd11D3 CU A CACAAAUC (34)       G A UUUdTdT (31)11 3 LacZ:Nkd12D2 CU A CACAAAUCA (35)       G A UUUdTdT (31) 12 2LacZ:Nkd13D1 CU A CACAAAUCAG (36)       G A UUUdTdT (31) 13 1 LacZ:Nkd14CU A CACAAAUCAGC (37)       G A UUUdTdT (31) 14 0 * A indicates a lockednucleic acid A in each sense strand.

The dual fluorescence assay of Example 2 was used to measure knockdownactivity. FIG. 7 shows that an mdRNA having a gap of up to 6 nucleotideshas substantial activity and the position of the gap may affect thepotency of knockdown. Thus, mdRNA having various sizes gaps that are invarious different positions and in different mdRNA sequences haveknockdown activity.

Example 6 Knockdown Activity of Substituted mdRNA

The activity of an influenza dsRNA RISC sequences having a nicked sensestrand and the sense strands having locked nucleic acid substitutionswere examined. The influenza RISC sequence G1498 of Example 2 wasgenerated with a sense strand having a nick at positions 8 to 14counting from the 5′-end. Each sense strand was substituted with one ortwo locked nucleic acids as shown in Table 5. The Qneg and Plasmid arenegative controls. Each of the mdRNAs was tested at a concentration of 5nM. The antisense strand used was 5′-CUCCGAAGAAAUAAGAUCCdTdT (SEQ IDNO:8).

TABLE 5 5′ Sense* 3′ Sense* Nick % mdRNA (SEQ ID NO.) (SEQ ID NO.) PosKD G1498-wt GGAUCUUAUUUCUUCGGAGdTdT (7) — — 85.8 G1498-L G GAUCUUAUUUCUUC G GAGdTdT (61) — — 86.8 G1498:Nkd8-1 G G AUCUUA (12)UUUCUUC G GAGdTdT (47)  8 36.0 G1498:Nkd8-2 G G AUCUU A  (40) U UUCUUC GGAGdTdT (54)  8 66.2 G1498:Nkd9-1 G G AUCUUAU (20)  UUCUUC GGAGdTdT (48)  9 60.9 G1498:Nkd9-2 G G AUCUUA U  (41)   U UCUUC GGAGdTdT (55)  9 64.4 G1498:Nkd10-1 G G AUCUUAUU (21)   UCUUC GGAGdTdT (49) 10 58.2 G1498:Nkd10-2 G G AUCUUAU U  (42)    U CUUC GGAGdTdT (56) 10 68.5 G1498:Nkd11-1 G G AUCUUAUUU (22)    CUUC GGAGdTdT (50) 11 75.9 G1498:Nkd11-2 G G AUCUUAUU U  (43)     C UUC GGAGdTdT (57) 11 67.1 G1498:Nkd12-1 G G AUCUUAUUUC (23)     UUC GGAGdTdT (51) 12 59.9 G1498:Nkd12-2 G G AUCUUAUUU C  (44)      U UC GGAGdTdT (58) 12 72.8 G1498:Nkd13-1 G G AUCUUAUUUCU (38)      UC GGAGdTdT (52) 13 37.1 G1498:Nkd13-2 G G AUCUUAUUUC U  (45)       U C GGAGdTdT (59) 13 74.3 G1498:Nkd14-1 G G AUCUUAUUUCUU (39)       C GGAGdTdT (53) 14 29.0 G1498:Nkd14-2 G G AUCUUAUUUCU U  (46)        CGGAGdTdT (60) 14 60.2 Qneg — — — 0 Plasmid — — — 3.6 *Nucleotides thatare bold and underlined are locked nucleic acids.

The dual fluorescence assay of Example 2 was used to measure knockdownactivity. These data show that increasing the number of locked nucleicacid substitutions tends to increase activity of an mdRNA having a nickat any of a number of positions. The single locked nucleic acid persense strand is most active when the nick is at position 11 (see FIG.8). But, multiple locked nucleic acids on each sense strand make mdRNAhaving a nick at any position as active as the most optimal nickposition with a single substitution (i.e., position 11) (FIG. 8). Thus,mdRNA having duplex stabilizing modifications make mdRNA essentiallyequally active regardless of the nick position.

Similar results were observed when locked nucleic acid substitutionswere made in the LacZ dicer substrate mdRNA of Example 1 (SEQ ID NOS:3and 4). The lacZ dicer was nicked at positions 8 to 14, and a duplicateset of nicked LacZ dicer molecules were made with the exception that theA at position 3 (from the 5′-end) of the 5′ sense strand was substitutedfor a locked nucleic acid A (LNA-A). As is evident from FIG. 11, most ofthe nicked lacZ dicer molecules containing LNA-A were as potent inknockdown activity as the unsubstituted lacZ dicer (see FIG. 9).

Example 7 mdRNA Knockdown of Influenza Virus Titer

The activity of a dicer substrate nicked dsRNA in reducing influenzavirus titer as compared to a wild-type dsRNA (i.e., not having a nick)was examined. The influenza dicer substrate sequence (25/27) is asfollows:

(SEQ ID NO: 62) Sense 5′-GGAUCUUAUUUCUUCGGAGACAAdTdG (SEQ ID NO: 11)Antisense 5′-CAUUGUCUCCGAAGAAAUAAGAUCCUUThese mdRNA sequences are nicked after position 12, 13, and 14,respectively, as counted from the 5′-end, and each sense strand also hasa G at position 2 substituted with locked nucleic acid G.

For the viral infectivity assay, Vero cells were seeded at 6.5×10⁴cells/well the day before transfection in 500 μl 10% FBS/DMEM media perwell. Samples of 100, 10, 1, 0.1, and 0.01 nM stock of each dsRNA werecomplexed with 1.0 μl (1 mg/ml stock) of Lipofectamine™ 2000(Invitrogen, Carlsbad, Calif.) and incubated for 20 minutes at roomtemperature in 150 μl OPTIMEM (total volume) (Gibco, Carlsbad, Calif.).Vero cells were washed with OPTIMEM, and 150 μl of the transfectioncomplex in OPTIMEM was then added to each well containing 150 μl ofOPTIMEM media. Triplicate wells were tested for each condition. Anadditional control well with no transfection condition was prepared.Three hours post transfection, the media was removed. Each well waswashed once with 200 μl PBS containing 0.3% BSA and 10 mM HEPES/PS.Cells in each well were infected with WSN strain of influenza virus atan MOI 0.01 in 200 μl of infection media containing 0.3% BSA/10 mMHEPES/PS and 4 μg/ml trypsin. The plate was incubated for 1 hour at 37°C. Unadsorbed virus was washed off with the 200 μl of infection mediaand discarded, then 400 μl DMEM containing 0.3% BSA/10 mM HEPES/PS and 4μg/ml trypsin was added to each well. The plate was incubated at 37° C.,5% CO₂ for 48 hours, then 50 μl supernatant from each well was tested induplicate by TCID₅₀ assays (Tissue-Culture Infective Dose 50, WHOprotocol) in MDCK cells and titers were estimated using the Spearman andKarber formula.

The results show that all of the G1498 nicked mdRNAs caused a 10-foldreduction in influenza viral titers (FIG. 10). That is, these mdRNAsshow about a 50% to 60% viral titer knockdown, even at a concentrationas low as 10 pM (FIG. 11).

An in vivo influenza mouse model was also used to examine the activityof a dicer substrate nicked dsRNA in reducing influenza virus titer ascompared to a wild-type dsRNA (i.e., not having a nick). Female BALB/cmice (age 8-10 weeks with 5-10 mice per group) were dosed intranasallywith 120 nmol/kg/day dsRNA (formulated inC12-norArg(NH₃+Cl—)-C12/DSPE-PEG2000/DSPC/cholesterol at a ratio of30:1:20:49) for three consecutive days before intranasal challenge withinfluenza strain PR8 (20 PFU/mouse). Two days after infection, wholelungs are harvested from each mouse and placed in a solution of PBS/0.3%BSA with antibiotics, homogenize, and measure the viral titer (TCID₅₀).Doses were well tolerated by the mice, indicated by less than 2% bodyweight reduction in any of the dose groups. The mdRNAs tested exhibitsimilar, if not slightly greater, virus reduction in vivo as compared tounmodified and unnicked G1498 dicer substrate (see FIG. 12). Hence,mdRNA are active in vivo.

Example 8 Effect of mdRNA on Cytokine Induction

The effect of the mdRNA structure on cytokine induction in vivo wasexamined Female BALB/c mice (age 7-9 weeks) were dosed intranasally withabout 50 μM dsRNA (formulated inC12-norArg(NH₃+Cl+C12/DSPE-PEG2000/DSPC/cholesterol at a ratio of30:1:20:49) or with 605 nmol/kg/day naked dsRNA for three consecutivedays. About four hours after the final dose is administered, the micewere sacrificed to collect bronchoalveolar fluid (BALF), and collectedblood is processed to serum for evaluation of the cytokine response.Bronchial lavage was performed with 0.5 mL ice-cold 0.3% BSA in salinetwo times for a total of 1 mL. BALF was spun and supernatants collectedand frozen until cytokine analysis. Blood was collected from the venacava immediately following euthanasia, placed into serum separatortubes, and allowed to clot at room temperature for at least 20 minutes.The samples were processed to serum, aliquoted into Millipore ULTRAFREE0.22 nm filter tubes, spun at 12,000 rpm, frozen on dry ice, and thenstored at −70° C. until analysis. Cytokine analysis of BALF and plasmawere performed using the Procarta™ mouse 10-Plex Cytokine Assay Kit(Panomics, Fremont, Calif.) on a Bio-Plex™ array reader. Toxicityparameters were also measured, including body weights prior to the firstdose on day 0 and again on day 3, just prior to euthanasia. Spleens wereharvested, weighed, and weights were normalized to final body weights.The results are provided in Table 6.

TABLE 6 In vivo Cytokine Induction by Naked mdRNA G1498:Nkd G1498:DSNkdG1498:DSNkd G1498:DSNkd Cytokine G1498 11-1 G1498:DS 12-1 13-1 14-1 IL-6Conc  90.68 10.07  77.35 17.17 18.21 38.59 (pg/mL) Fold decrease — 9 — 54 2 IL-12 Conc 661.48 20.32 1403.61  25.07 37.70 57.02 (p40) (pg/mL)Fold decrease — 33 — 56 37 25 TNFα Conc 264.49 25.59 112.95 20.52 29.0064.93 (pg/mL) Fold decrease — 10 — 6 4 2

The mdRNA (RISC or dicer sized) induced cytokines to lesser extent thanthe intact (i.e., not nicked) parent molecules. The decrease in cytokineinduction was greatest when looking at IL-12(p40), the cytokine withconsistently the highest levels of induction of the 10 cytokinemultiplex assay. For the mdRNA, the decrease in IL-12 (p40) ranges from25- to 56-fold, while the reduction in either IL-6 or TNFα induction wasmore modest (the decrease in these two cytokines ranges from 2- to10-fold). Thus, the mdRNA structure appears to provide an advantage invivo in that cytokine induction is minimized compared to unmodifieddsRNA.

Similar results were obtained with the formulated mdRNA, although thereduction in induction was not as prominent. In addition, the presenceor absence of a locked nucleic acid has no effect on cytokine induction.These results are shown in Table 7.

TABLE 7 In vivo Cytokine Induction by Formulated mdRNA G1498:NkdG1498:Nkd G1498:DSNkd G1498:DSNkd Cytokine G1498:DS 12-1 13-1 14-1 13IL-6 Conc (pg/mL) 29.04 52.95 10.28 7.79 44.29 Fold decrease — −1.8 3 4−1.5 IL-12 (p40) Conc (pg/mL) 298.93  604.24 136.45 126.71 551.49 Folddecrease — 0 2 2 1 TNFα Conc (pg/mL) 13.49 21.35 3.15 3.15 18.69 Folddecrease — −1.6 4 4 1.4

The teachings of all of references cited herein including patents,patent applications and journal articles are incorporated herein intheir entirety by reference. Although the foregoing disclosure has beendescribed in detail by way of example for purposes of clarity ofunderstanding, it will be apparent to the artisan that certain changesand modifications may be practiced within the scope of the appendedclaims which are presented by way of illustration not limitation. Inthis context, various publications and other references have been citedwithin the foregoing disclosure for economy of description. It is noted,however, that the various publications discussed herein are incorporatedsolely for their disclosure prior to the filing date of the presentapplication, and the inventors reserve the right to antedate suchdisclosure by virtue of prior invention.

1.-64. (canceled)
 65. A method for reducing expression of a target genein a cell, said method comprising: contacting a target gene expressingcell with a composition comprising a 19 to 30 base pair double-strandedribonucleic acid, wherein said target gene expression is reduced as aconsequence of contacting said cell with said composition, saidribonucleic acid comprising (a) an A strand having a length of 15 to 30nucleotides and a nucleotide sequence that is complementary to anucleotide sequence in said target gene; (b) an S1 strand having alength of 5 to 25 nucleotides, wherein said S1 strand anneals to said Astrand thereby forming a first double-stranded region of 5 to 15 basepairs; and (c) an S2 strand having a length of 5 to 25 nucleotides,wherein said S2 strand anneals to said A strand thereby forming a seconddouble-stranded region of 3 to 25 base pairs and wherein said annealedS2 strand is separated from said annealed S1 strand by a nick or a gap.66. The method of claim 65 wherein said composition comprises aliposome, a hydrogel, a cyclodextrin, a biodegradable nanocapsule, abioadhesive microsphere, or a proteinaceous vector.
 67. The method ofclaim 66 wherein said liposome is a surface-modified liposome.
 68. Themethod of claim 67 wherein said liposome comprises one or more lipidsselected from the group consisting of a non-cationic lipid and acationic lipid.
 69. The method of claim 65 wherein said double-strandedribonucleic acid is combined, complexed, or conjugated with a peptide.70. The method of claim 69 wherein said peptide facilitates delivery ofsaid double-stranded ribonucleic acid into said target cell.
 71. Themethod of claim 69 wherein said peptide is selected from the groupconsisting of PN27, PN28, PN29, PN58, PN61, PN73, PN158, PN159, PN173,PN182, PN202, PN204, PN250, PN361, PN365, PN404, PN453, and PN509. 72.The method of claim 69 wherein said peptide comprises an N-terminalprotein transduction domain from HIV TAT.
 73. The method of claim 65wherein overexpression or inappropriate expression of said target geneis associated with one or more disease or disorder.
 74. The method ofclaim 73 wherein said disease or disorder is selected from the groupconsisting of a hyperproliferative disease or disorder, an angiogenicdisease or disorder, a metabolic disease or disorder, and aninflammatory disease or disorder.
 75. The method of claim 73 whereinsaid disease or disorder is selected from the group consisting of acardiac disease, a pulmonary disease, a neovascularization, an ischemia,age-related macular degeneration, diabetic retinopathy,glomerulonephritis, diabetes, asthma, chronic obstructive pulmonarydisease, chronic bronchitis, lymphangiogenesis, and atherosclerosis. 76.The method of claim 74 wherein said hyperproliferative disease ordisorder is selected from the group consisting of a neoplasm, acarcinoma, a sarcoma, a tumor, and a cancer.
 77. The method of claim 74wherein said hyperproliferative disease or disorder is selected from thegroup consisting of an oral cancer, a throat cancer, a laryngeal cancer,an esophageal cancer, a pharyngeal cancer, a nasopharyngeal cancer, anoropharyngeal cancer, a gastrointestinal tract cancer, agastrointestinal stromal tumor (GIST), a small intestine cancer, a coloncancer, a rectal cancer, a colorectal cancer, an anal cancer, apancreatic cancer, a breast cancer, a cervical cancer, uterine cancer, avulvar cancer, vaginal cancer, urinary tract cancer, bladder cancer,kidney cancer, adrenocortical cancer, islet cell carcinoma, gallbladdercancer, stomach cancer, prostate cancer, ovarian cancer, endometrialcancer, trophoblastic tumor, testicular cancer, penial cancer, bonecancer, osteosarcoma, liver cancer, extrahepatic bile duct cancer, skincancer, basal cell carcinoma (BCC), lung cancer, small cell lung cancer,non-small cell lung cancer (NSCLC), brain cancer, melanoma, Kaposi'ssarcoma, eye cancer, head and neck cancer, squamous cell carcinoma ofhead and neck, tymoma, thymic carcinoma, thyroid cancer, parathyroidcancer, Rippel-Linda syndrome, leukemia, acute myeloid leukemia, chronicmyelogenous leukemia, acute lymphoblastic leukemia, hairy cell leukemia,lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, T-cell lymphoma,multiple myeloma, malignant pleural mesothelioma, Barrett'sadenocarcinoma, and Wilm's tumor.
 78. The method of claim 74 whereinsaid inflammatory disease or disorder is selected from the groupconsisting of diabetes mellitus, rheumatoid arthritis, pannus growth ininflamed synovial lining, collagen-induced arthritis, spondylarthritis,ankylosing spondylitis, multiple sclerosis, encephalomyelitis,inflammatory bowel disease, Chron's disease, psoriasis or psoriaticarthritis, myasthenia gravis, systemic lupus erythematosis,graft-versus-host disease, atherosclerosis, and an allergy.